Amnion-derived cell compositions, methods of making and uses thereof

ABSTRACT

The invention is directed to substantially purified amnion-derived cell populations, compositions comprising the substantially purified amnion-derived cell populations, and to methods of creating such substantially purified amnion-derived cell populations, as well as methods of use. The invention is further directed to antibodies, in particular, monoclonal antibodies, that bind to amnion-derived cells or, alternatively, to one or more amnion-derived cell surface protein markers. The invention is further directed to methods for producing the antibodies, methods for using the antibodies, and kits comprising the antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Utility applicationSer. No. 11/392,892, filed Mar. 29, 2006, which claims priority under 35USC §119(e) to U.S. Provisional Application No. 60/666,949, filed Mar.31, 2005, U.S. Provisional Application No. 60/699,257, filed Jul. 14,2005, U.S. Provisional Application No. 60/742,067, filed Dec. 2, 2005,and under 35 USC §120 to U.S. Utility application Ser. No. 11/333,849,filed Jan. 18, 2006, the contents of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The field of the invention is directed to amnion-derived cellpopulations, compositions comprising the amnion-derived cellpopulations, expanded amnion-derived cell populations, methods ofcreating such amnion-derived cell populations, as well as methods ofuse. The field is also directed to antibodies, in particular, monoclonalantibodies, that bind to amnion-derived cells or, alternatively, to oneor more amnion-derived cell surface protein markers, methods forproducing the antibodies, methods for using the antibodies, and kitscomprising the antibodies. The field of the invention is furtherdirected to novel pancreatic cell compositions, methods for theirproduction and uses thereof, and to novel cell culture factor systems.

DESCRIPTION OF RELATED ART

Preliminary evidence suggests that amnion epithelial cells isolated andplaced in culture exhibit many of the characteristics necessary todefine a stem cell population (Brivanlou, A. H., et al., Science, 2003.300(5621): p. 913-6).

Placental-derived stem cells isolated from placenta have been shown toexhibit heterogeneous protein expression of the stage-specific embryonicantigens SSEA-3 and SSEA-4, TRA 1-60, TRA 1-81, c-kit, and Thy-1 (seeUS2003/0235563 and US2004/0161419). These cells have also been shown toexpress the cell surface proteins Oct-4 and nanog, markers reportedlyexpressed by pluripotent stem cells. Under appropriate conditions,placental-derived stem cells have been shown to differentiate into cellswith characteristics of liver cells (hepatocytes), pancreatic cells(i.e. alpha and beta cells), central nervous system cells (neurons andglia), cardiac muscle cells (cardiomyocytes) and vascular endothelialcells. Placental-derived stem cells are non-tumorigenic upontransplantation (Miki, T., et al., Stem Cells 2005; 23:1549-1559). Infact, tumors have not been observed in immuno-compromised mice followingtransplantation of more than 20 million placental-derived stem cells,conditions under which ES cells form non-malignant tumors known asteratomas. US2003/0235563 and US2004/0161419 disclose preliminarystudies indicating that placental-derived stem cells cultured inMatrigel supplemented with 10 mM nicotinamide for 14 days expressinsulin and glucagon as well as the pancreatic cell markers PDX1(faint), Pax6 and Nkx2.2.

Others have transplanted amniotic cells into volunteers and patients inan attempt to correct lysosomal storage diseases with no evidence oftumorigenicity (Tylki-Szymanska, A., et al., Journal of InheritedMetabolic Disease, 1985. 8(3): p. 101-4; Yeager, A. M., et al., AmericanJournal of Medical Genetics, 1985. 22(2): p. 347-55).

Amniotic membrane is regularly transplanted as a graft for ocularsurface reconstruction without subsequent tumor formation (John, T.,Human amniotic membrane transplantation: past, present, and future.Opthalmol Clin North Am, 2003. Mar. 16(1): p. 43-65, vi.). This lack oftumorigenicity is an important distinction between ES cells andplacental-derived stem cells.

Results of preliminary studies with other cells are disclosed in WO2005/017117, WO2005/0042595, US 2005/0019865, US2005/0032209,US2005/0037491, US2005/0058631, and US2005/0054093. Results ofpreliminary studies with these other cells indicate that they have thepotential to differentiate into various cell types.

Amniotic membranes have been used clinically as wound dressing for burnpatients for over 100 years to promote epithelialization, reduce pain,and prevent infection (Bose, B. (1979) Ann R Coll Surg Engl, 61:444-7;Sawhney, C. P. (1989) Burns, 15:339-42, Thomson, P. D., Parks, D. H.(1981) Ann Plast Surg, 7:354-6). US2003/0235580 describes a method ofdelivering therapeutic molecules to skin using amniotic epithelialcells. US2004/0057938 describes the use of a human amniotic membranecomposition for prophylaxis and treatment of diseases and conditions ofthe eye and skin. U.S. Pat. No. 4,361,552 describes a method of treatinga wound or burn, which comprises covering the surface of the wound orburn with a cross-linked amnion dressing.

US2004/0170615 describes the use of compounds expressed in fetal tissuefor use in skin repair and the improvement of skin appearance.

Wei, et al, (Wei, J P, et al, (2003) Cell Transplantation 12:545-552)have shown that human amnion-isolated cells can normalize blood glucosein streptozotocin-induced diabetic mice.

BACKGROUND OF THE INVENTION

Stem Cells—Stem cells have the remarkable potential to develop into manydifferent cell types in the body. Serving as a repair system for thebody, they can theoretically divide without limit to replenish othercells throughout a person's life. When a stem cell divides, each newcell has the potential to either remain a stem cell or become anothertype of cell with a more specialized function, such as a muscle cell, ared blood cell, or a brain cell. Perhaps the most important potentialapplication of human stem cells is the generation of cells and tissuesthat could be used for cell-based therapies. Examples of stem cellstudies are provided (Tylki-Szymanska, A., et al., Journal of InheritedMetabolic Disease, 1985. 8(3): p. 101-4; Yeager, A. M., et al., AmericanJournal of Medical Genetics, 1985. 22(2): p. 347-55; John, T., 2003.16(1): p. 43-65, vi.).

Placental tissue is abundantly available as a discarded source of a typeof stem cell called placental-derived stem cells. Although discarded aspart of the placental membranes, lineage analysis shows that unlikeother tissues of the placenta, the epithelial layer of the amnion, fromwhich the placental-derived stem cells are isolated, is uniquelydescended from the epiblast in embryonic development (FIG. 1). Theepiblast contains the cells that will ultimately differentiate into theembryo and cells that will give rise to an extraembryonic tissue, theamnion. Thus far, only four cell types that have been described in theliterature as being pluripotent. These are the inner cell mass (ICM) ofthe pre-implantation embryo, which gives rise to the epiblast, theepiblast itself, embryonic stem (ES) and embryonic germ cells (EG).Thus, identification, purification and propagation of a pluripotent cellpopulation from discarded amnion tissue would provide an extremelyvaluable source of stem cells for replacement cell therapy.

With an average yield of over 200 million placental-derived stem cellsper placenta, large numbers of cells are available from this source. Ifplacental-derived stem cells were to become useful cells fortransplantation medicine, they could provide a nearly inexhaustiblesupply of starting material in every part of the world. No other stemcell source provides such a large starting population of cells, andcollection does not require an invasive or destructive procedure.Furthermore, there are no ethical, religious or social issues associatedwith these placental-derived stem cells as the tissue is derived fromthe placenta.

Another important consideration in stem cell therapies is grafttolerance. In humans, the protein expression of the cell surface markerHLA-G was originally thought to be restricted to immune-privileged sitessuch as placenta, as well as related cells, including some isolated fromamniotic fluid, placental macrophages, and cord blood, thus implicatingits role in maternal-fetal tolerance (Urosevic, M. and Dummer, R. (2002)ASHI Quarterly; 3rd Quarter 2002:106-109). Additionally, studiesinvolving heart-graft acceptance have suggested that the proteinexpression of HLA-G may enhance graft tolerance (Lila, N., et al. (2000)Lancet 355:2138; Lila, N. et al. (2002) Circulation 105:1949-1954).HLA-G protein is not expressed on the surface of undifferentiated ordifferentiated embryonic stem cells (Drukker, M, et al. (2002) PNAS99(15):9864-9869). Thus, it is desirable that stems cells intended forcell-based therapies express HLA-G protein.

Wound Healing—Placental-derived cells have been shown to secrete manycytokines and growth factors including prostaglandin E2, PGES, TGF-β,EGF, IL-4, IL-8, TNF, interferons, activin A, noggin, bFGF, someneuroprotective factors, and many angiogenic factors (Koyano et al.,(2002) Develop. Growth Differ. 44:103-112; Blumenstein et al. (2000)Placenta 21:210-217; Tahara et al. (1995) J. Clin. Endocrinol. Metabol.80:138-146; Paradowska et al. (1997) Placenta 12:441-446; Denison et al.(1998) Hum. Reprod. 13:3560-3565; Keelan et al. (1998) Placenta19:429-434; Uchida et al. (2000) J. Neurosci. Res. 62:585-590; Sun etal. (2003) J. Clin. Endocrinol. Metabol. 88(11):5564-5571; Marvin et al.(2002) Am. J. Obstet. Gynecol. 187(3):728-734). Many of these cytokinesare associated with wound healing and some have been credited withcontributing to scarless healing in the fetus.

Approximately 50 million surgical procedures are performed in the UnitedStates each year. An additional 50 million wounds result from traumaticinjuries. Subsequent acute wound healing failure at any anatomic siteresults in increased morbidity and mortality. Non-limiting examples ofacute wound failure include muscle, fascial and skin dehiscence,incisional hernia formation, gastrointestinal fistulization and vascularanastamotic leaks. Besides the immediate functional disability, acutewounds that fail usually go on to form disabling scars.

Incisional hernias of the abdominal wall provide an excellent paradigmto study the mechanism and outcome of acute wound healing failure.Large, prospective, well-controlled series have shown that 11-20% ofover 4 million abdominal wall fascial closures fail leading to ventralincisional hernia formation. Even after repair of acute wound failure,recurrence rates remain as high as 58%. Improvements in suture material,stitch interval, stitch distance from the margin of the wound, andadministration of prophylactic antibiotics to avoid infectionsignificantly decreased the rates of clinically obvious acute wounddehiscence, but only led to small decreases in the rates of ventralhernia formation and recurrence. The introduction of tissue prostheses,typically synthetic meshes, to create a tension-free bridge or patch ofthe myo-fascial defect reduced first recurrence rates significantly,supporting the concept that mechanical factors predominate in thepathogenesis of recurrent hernia.

Traditional surgical teaching is that laparotomy wound failure is a rareevent, with reported “fascial dehiscence” rates clustered around 0.1%.One prospective study found that the true rate of laparotomy woundfailure is closer to 11%, and that the majority of these (94%) go on toform incisional hernias during the first three years after abdominaloperations. This is more in line with the high incidence of incisionalhernia formation. The real laparotomy wound failure rate is therefore100 times what most surgeons think it is. In simplest terms, mostincisional hernias are derived from clinically occult laparotomy woundfailures, or occult fascial dehiscences. The overlying skin wound heals,concealing the underlying myofascial defect. This mechanism of earlymechanical laparotomy wound failure is more consistent with modern acutewound healing science. There are no other models of acute wound healingsuggesting that a successfully healed acute wound goes on to breakdownand mechanically fail at a later date. This mechanism is also unique inthat it assumes that the majority of abdominal wall laparotomy woundfailures occur in hosts with no clearly identifiable wound healingdefect. One model of laparotomy wound failure that was developedresulted in incisional hernias. The paramedian skin flap design isolatesthe skin and myofascial incisions and allows one to simultaneously studymidline laparotomy wound repair and paramedian dermal repair. Skin andmyofascial repairs can be controlled to achieve 100% intact repairs, or100% structural failure and wound dehiscence.

Cosmetics—Fetal skin has much more effective repair mechanisms, and,once wounded, it is able to heal without the formation of scars. Thiscapability does appear to require the fetal immune system, fetal serum,or amniotic fluid (Bleacher J C, et al., J Pediatr Surg 28: 1312-4,1993); Ihara S, Motobayashi Y., Development 114: 573-82. 1992). Suchabilities of fetal tissue have led to the suggested use of compoundsproduced by fetal tissue for regenerating and/or improving theappearance of skin (see, for example, US 2004/0170615, which isincorporated by reference in its entirety herein).

Diabetes—Traditional insulin therapy prolongs the life of a patient withType I diabetes but does not prevent the long-term systemiccomplications that arise as the disease progresses. Even the bestinjection/infusion regime to monitor and control systemic glucose levelswithin an acceptable range inevitably leads to a deterioration of tissuemicrovascularization resulting in the plethora of health-relatedcomplications associated with the disease. These complications can beattributed to the inability of injectable or orally administered insulinto completely substitute for the insulin secretion from a normalcomplement of pancreatic islets. The failure of insulin as a substitutefor the pancreatic islet beta cell can largely be explained when oneexamines the cellular architecture of a pancreatic islet itself.Intensive inter-cellular regulation of hormone secretion, accomplishedby immediate islet cell proximity, is necessary to prevent the largetemporal fluctuations in blood glucose levels that are responsible forcellular damage and the ensuing complications of the disease.

Presently, transplantation of cadaver pancreas or isolation andtransplantation of cadaver islets are the only alternative treatments toinsulin administration that exist for patients dependent on insulin tocontrol their diabetes. The scarcity of donor tissue reserves thesealternative therapies for select patients that are unable to stabilizetheir blood glucose adequately using traditional insulininjection/infusion regimes.

This conundrum profiles diabetes as a prime candidate for cell-basedtherapies. This candidacy is made stronger by the unique quality ofislets to function as self-contained, functional, glucose-sensingmulticellular units

Studies have also been undertaken to promote differentiation of stemcells, progenitor cells or their progeny using protein transductiondomains (PTDs) such as that contained in the HIV-1 TAT protein. TheHIV-1 TAT protein has been found to penetrate cells in aconcentration-dependent, receptor-independent fashion. Studies have beenundertaken with TAT PTDs to determine their usefulness in deliveringproteins to cells (see, for example, US 2005/0048629, Wadia et al.,2004, Nature Medicine 10:310-315 and Krosl et al., 2003, Nature Medicine9:1-10). Such proteins may be used to promote differentiation of stemcells, progenitor cells or their progeny.

BRIEF SUMMARY OF THE INVENTION

Although heterogeneous populations of placental-derived stem cells havebeen previously characterized using established embryonic stem cellsurface protein markers such as c-kit, SSEA-3, and SSEA-4, a set ofprotein markers useful for characterizing and isolating a preferredsubstantially purified population of cells is required. Thissubstantially purified population of cells, termed amnion-derived cells,could then be fully discriminated from other cells such as embryonicstem cells, mesenchymal stem cells or adult-derived stem cells.Therefore, it is an object of this invention to provide such proteinmarkers capable of characterizing and isolating amnion-derived cellsfrom placental-derived stem cells. It is also an object of the inventionto use those protein markers as antigens to make hybridoma cell linesthat produce monoclonal antibodies specific for those protein markers.

Accordingly, a first aspect of the invention is a substantially purifiedpopulation of amnion-derived cells that is negative for expression ofthe protein markers CD90 and CD117.

A second aspect of the invention is a substantially purified populationof the first aspect of the invention that is further negative forexpression of the protein marker CD105.

A third aspect of the invention is a substantially purified populationof the first aspect of the invention that is positive for expression ofthe protein marker CD29.

A fourth aspect of the invention is a substantially purified populationof the third aspect of the invention that is negative for expression ofthe protein marker CD105.

A fifth aspect of the invention is a substantially purified populationof the third aspect of the invention, that is further positive forexpression of at least one of the protein markers selected from thegroup consisting of CD9, CD10, CD26, CD71, CD166, CD227, EGF-R, SSEA-4,and HLA-G.

A sixth aspect of the invention is a substantially purified populationof the fourth aspect of the invention, that is further positive forexpression of at least one of the protein markers selected from thegroup consisting of CD9, CD10, CD26, CD71, CD166, CD227, EGF-R, SSEA-4,and HLA-G.

A seventh aspect of the invention is a substantially purified populationof the second aspect of the invention that is further negative for theexpression of at least one of the protein markers selected from thegroup consisting of CD140b, telomerase, CD34, CD44, and CD45.

An eighth aspect of the invention is a substantially purified populationof the fourth aspect of the invention that is further negative for theexpression of at least one of the protein markers selected from thegroup consisting of CD140b, telomerase, CD34, CD44, and CD45.

A ninth aspect of the invention is a substantially purified populationof the sixth aspect of the invention that is further negative for theexpression of at least one of the protein markers selected from thegroup consisting of CD140b, telomerase, CD34, CD44, and CD45.

A tenth aspect of the invention is a population of amnion-derived cellsof aspects one through nine of the invention, which is a composition. Ina preferred embodiment, the composition is a pharmaceutical composition.

An eleventh aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the firstaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the population of amnion-derivedcells with anti-CD90 and anti-CD117 antibodies; and c) separating theamnion-derived cells that bind to the antibodies from the cells that donot bind to the antibodies such that the substantially purifiedpopulation of amnion-derived cells of the first aspect of the inventionthat do not bind to the antibodies is obtained.

A twelfth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the secondaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with anti-CD90,anti-CD117, and anti-CD105 antibodies; and c) separating the cells thatbind to the antibodies from the cells that do not bind to the antibodiessuch that the substantially purified population of amnion-derived cellsof the second aspect of the invention that do not bind to the antibodiesis obtained.

A thirteenth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the seventhaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) anti-CD90,anti-CD117, and anti-CD105 antibodies and (ii) with at least oneantibody selected from the group consisting of anti-CD140b, anti-CD34,anti-CD44, and anti-CD45 antibodies; and c) separating the cells thatbind to the antibodies of (i) from the cells that do not bind to theantibodies of (i) and separating the cells that bind to the antibodiesof (ii) from the cells that do not bind to the antibodies of (ii); andsuch that the substantially purified population of amnion-derived cellsof the seventh aspect of the invention that do not bind to theantibodies of (i) and (ii) is obtained.

A fourteenth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the thirdaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) anti-CD90 andanti-CD117 antibodies and (ii) with an anti-CD29 antibody; and c)separating the cells that do not bind to the antibodies of (i) from thecells that do bind to the antibody of (i) and separating the cells thatdo not bind to the antibodies of (ii) from the cells that do bind to theantibody of (ii) such that the substantially purified population ofamnion-derived cells of the third aspect of the invention that do notbind to the antibodies of (i) and do bind to the antibody of (ii) isobtained.

A fifteenth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the fourthaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) anti-CD90,anti-CD117 and anti-CD105 antibodies and (ii) with an anti-CD29antibody; and c) separating the cells that do not bind to the antibodiesof (i) from the cells that do bind to the antibodies of (i) andseparating the cells that do not bind to the antibody of (ii) from thecells that do bind to the antibody of (ii) such that the substantiallypurified population of amnion-derived cells of the fourth aspect of theinvention that do not bind to the antibodies of (i) and do bind to theantibody of (ii) is obtained.

A sixteenth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the fifthaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) anti-CD90,anti-CD117 antibodies and (ii) anti-CD29 antibodies and (iii) with oneor more antibodies selected from the group consisting of anti-CD9,anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R,anti-SSEA-4, and anti-HLA-G antibodies; and c) separating the cells thatdo not bind to the antibody of (i) from the cells that do bind to theantibodies of (i) and separating the cells that do not bind to theantibody of (ii) from the cells that do bind to the antibody of (ii) andseparating the cells that do not bind to the antibodies of (iii) fromthe cells that do bind to the antibodies of (iii) such that thesubstantially purified population of amnion-derived cells of the fifthaspect of the invention that do not bind to antibodies of (i), do bindto antibody of (ii) and do bind to antibodies of (iii) is obtained.

A seventeenth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the sixthaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) anti-CD90,anti-CD117, and anti-CD105 antibodies and (ii) and anti-CD29 antibodiesand (iii) with one or more antibodies selected from the group consistingof anti-CD9, anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227,anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies; and c) separatingthe cells that do not bind to the antibody of (i) from the cells that dobind to the antibodies of (i) and separating the cells that do not bindto the antibody of (ii) from the cells that do bind to the antibody of(ii) and separating the cells that do not bind to the antibodies of(iii) from the cells that do bind to the antibodies of (iii) such thatthe substantially purified population of amnion-derived cells of thesixth aspect of the invention that do not bind to antibodies of (i), dobind to antibody of (ii) and do bind to antibodies of (iii) is obtained.

An eighteenth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the eighthaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) anti-CD90,anti-CD117, and anti-CD105 antibodies and (ii) and anti-CD29 antibodiesand (iii) with one or more antibodies selected from the group consistingof anti-CD140b, anti-CD34, anti-CD44, and anti-CD45 antibodies; and c)separating the cells that do not bind to the antibody of (i) from thecells that do bind to the antibodies of (i) and separating the cellsthat do not bind to the antibody of (ii) from the cells that do bind tothe antibody of (ii) and separating the cells that do not bind to theantibodies of (iii) from the cells that do bind to the antibodies of(iii) such that the substantially purified population of amnion-derivedcells of the eighth aspect of the invention that do not bind toantibodies of (i), do bind to antibody of (ii) and do not bind toantibodies of (iii) is obtained.

A nineteenth aspect of the invention is a method of obtaining thesubstantially purified population of amnion-derived cells of the ninthaspect of the invention, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) anti-CD90,anti-CD117, and anti-CD105 antibodies and (ii) and anti-CD29 antibodiesand (iii) one or more antibodies selected from the group consisting ofanti-CD140b, anti-CD34, anti-CD44, and anti-CD45 antibodies and (iv) oneor more antibodies selected from the group consisting of anti-CD9,anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R,anti-SSEA-4, and anti-HLA-G antibodies; and c) separating the cells thatdo not bind to the antibody of (i) from the cells that do bind to theantibodies of (i) and separating the cells that do not bind to theantibody of (ii) from the cells that do bind to the antibody of (ii) andseparating the cells that do not bind to the antibodies of (iii) fromthe cells that do bind to the antibodies of (iii) and separating thecells from that do bind to the antibody of (iv) from the cells that donot bind to the antibodies of (iv) such that the substantially purifiedpopulation of amnion-derived cells of the ninth aspect of the inventionthat do not bind to antibodies of (i), do bind to antibody of (ii), donot bind to antibodies of (iii) and do bind to the antibodies of (iv) isobtained.

A twentieth aspect of the invention is a method of obtaining asubstantially purified population of amnion-derived cells, comprising:a) providing a population of amnion-derived cells; b) contacting thecells with one or more antibodies selected from the group consisting ofanti-CD105, anti-CD90, anti-CD117, anti-CD140b, anti-CD34, anti-CD44,and anti-CD45 antibodies; and one or more antibodies selected from thegroup consisting of anti-CD29, anti-CD9, anti-CD10, anti-CD26,anti-CD71, anti-CD166, anti-CD227, anti-EGF-R, anti-SSEA-4, andanti-HLA-G antibodies; and c) separating the cells that do not bind tothe antibodies of (i) from the cells that do bind to the antibody of (i)and separating the cells that do not bind to the antibodies of (ii) fromthe cells that do bind to the antibody of (ii) such that a substantiallypurified population of amnion-derived cells that do not bind to theantibodies of (i) and do bind to the antibody of (ii) is obtained.

A twenty-first aspect of the invention is the method of aspects 11through 20, wherein the cells are separated by FACS sorting.

A twenty-second aspect of the invention is one in which the antibodiesof aspects 11 through 20 are monoclonal antibodies, fully humanantibodies, humanized antibodies, chimeric antibodies, a scfv, or afragment or derivative of any one of the aforementioned antibodies.

In addition to aspects 1 through 22 of the invention, additional aspectsprovide for expanded and/or clustered amnion-derived cells andpopulations, which provide several advantages over previously describedplacental-derived cell compositions as well as embryonic stem cellscompositions.

Accordingly, a twenty-third aspect of the invention is theamnion-derived cells of the first aspect of the invention, which are anexpanded amnion-derived cell composition. In a preferred embodiment, thecomposition of aspect twenty three is animal-free. In another preferredembodiment the composition is a clustered amnion-derived cellcomposition.

A twenty-fourth aspect of the invention is a composition comprisingconditioned medium obtained from the expanded amnion-derived cellcomposition of the twenty-third aspect of the invention.

A twenty-fifth aspect of the invention is a composition comprising celllysate obtained from the amnion-derived cell composition of thetwenty-third aspect of the invention.

A twenty-sixth aspect of the invention is the expanded amnion-derivedcell composition of the twenty-third aspect having a concentration of atleast 500×10⁶ amnion-derived cells/g of starting amnion.

A twenty-seventh aspect of the invention is a method of creating ahepatocyte comprising differentiating, in vitro or in vivo, anamnion-derived cell population of the first aspect of the invention.

A twenty-eighth aspect of the invention is a hepatocyte created by themethod of the twenty-seventh aspect of the invention.

A twenty-ninth aspect of the invention is a liver assist devicecomprising an amnion-derived cell composition of the twenty-seventhaspect of the invention.

A thirtieth aspect of the invention is a method of creating acardiomyocyte comprising differentiating, in vitro or in vivo, anamnion-derived cell population of the first aspect of the invention.

A thirty-first aspect of the invention is a cardiomyocyte created by themethod of the thirtieth aspect of the invention.

A thirty-second aspect of the invention is a method for promotingaccelerated wound healing in an injured patient in need thereofcomprising administering to the patient one or more compositions ofplacental-derived cells. In a preferred embodiment the composition ofplacental-derived cells is an expanded amnion-derived cell composition.In another preferred embodiment of the method the composition isadministered in a scaffold or matrix. In a specific, preferredembodiment, the scaffold or matrix is amniotic tissue. In anotherpreferred embodiment, the wound is selected from the group consisting ofmechanical, thermal, acute, chronic, infected, and sterile wounds. Andin yet another preferred embodiment the injured patient is a human.

A thirty-third aspect of the invention is a cosmetic preparationcomprising one or more compositions of placental-derived cells. In apreferred embodiment, the composition of placental-derived cells is anexpanded amnion-derived cell composition.

A thirty-fourth aspect of the invention is a method for treating hearingloss in a patient in need thereof comprising administering to thepatient one or more compositions of placental-derived cells. In apreferred embodiment, the composition of placental-derived cells is anexpanded amnion-derived cell composition.

A thirty-fifth aspect of the invention is a method of proliferatingembryonic stem cells comprising using the amnion-derived cells of thefirst aspect as a feeder layer. One preferred embodiment of this aspectis one which is free of animal products.

In addition to aspects 23 through 35 of the invention, the inventionalso contemplates compositions comprising differentiated amnion-derivedcell populations, method for identifying such populations, methods ofmaking such populations, and methods of using them.

Accordingly, a thirty-sixth aspect of the invention is the population ofthe first aspect of the invention wherein the cells express a pancreaticprogenitor cell marker protein. In a preferred embodiment, theprogenitor cell marker is PDX1 protein. In another preferred embodiment,the PDX1 protein is expressed in the nucleus.

A thirty-seventh aspect of the invention is the population of cells ofthe thirty-sixth aspect further optionally expressing any one or more ofthe protein markers selected from the group consisting of Foxa2, p48,Hblx9 and Neurogenin 3 (Ngn3). In a preferred embodiment, the cellsfurther optionally express any one or more of the protein markersselected from the group consisting of NKx2.2, Nkx6.1, insulin andislet-1.

In a thirty-eighth aspect of the invention is a population of cells ofthe thirty-sixth aspect, wherein the cells are differentiated pancreaticprogenitor cells. In a preferred embodiment, the differentiatedprogenitor cells express any one or more of the protein markers selectedfrom the group consisting of PDX1, insulin, C-peptide, somatostatin,pancreatic polypeptide, and glucagon. In another preferred embodimentthe differentiated pancreatic progenitor cells are islet-like cells. Ina specific, preferred embodiment the islet-like cells are alpha, beta,delta or phi cells and in a most preferred embodiment the islet-likecells are functional islet-like cells. In another preferred embodimentthe functionality of the islet-like cells is incrementalglucose-dependent insulin secretion.

A thirty-ninth aspect of the invention is an islet comprising thepopulation of cells of the thirty-sixth and thirty-eighth aspects.

A fortieth aspect of the invention is a tissue comprising the populationof the thirty-sixth and thirty-eighth aspects.

A forty-first aspect of the invention is the population of thethirty-sixth aspect wherein the cells form spheroids. In a preferredembodiment the spheroids form buds. In another preferred embodiment thebuds express PDX1 protein and in a most preferred embodiment the PDX1protein is expressed in the nucleus.

A forty-second aspect of the invention is the population of thethirty-sixth aspect of the invention which comprises one or moremammalian embryonic islet progenitor cells. In a preferred embodiment ofthis aspect, the mammalian embryonic islet progenitor cells are humancells.

A forty-third aspect of the invention is the population of thethirty-sixth aspect wherein the cells express a heterologous protein. Inone embodiment the heterologous protein is a TAT fusion protein. Inspecific embodiments the TAT fusion protein is TAT-PDX1, TAT-Hblx9,TAT-p48, TA-Ngn3 or TAT-Foxa2. In another preferred embodiment theheterologous protein is a therapeutic protein of interest.

A forty-fourth aspect of the invention is the population of thethirty-sixth aspect wherein the cells have the identifyingcharacteristics of endoderm. In a preferred embodiment the identifyingcharacteristics of endoderm are expression of HNF1α, HNF1β, HNF4α, HNF6,Foxa2 and PDX1 proteins. In another preferred embodiment the cellsfurther optionally express any one or more of the protein markers Sox17, Cerberus, Hesx 1, LeftyA, Otx 1 or Otx2.

A forty-fifth aspect of the invention is a composition comprising one ormore nuclei isolated from pancreatic progenitor cells of the thirtyeighth aspect, wherein the cells express PDX 1 protein in the nucleusand/or express Nkx2.2, Nkx6.1, insulin and islet-1 protein and/or havethe identifying characteristics of endoderm. In a preferred embodimentof this aspect the identifying characteristics of endoderm are proteinexpression of HNF1α, HNF1β, HNF4α, HNF6, Foxa2 and PDX 1. In anotherpreferred embodiment the cells further optionally express any one ormore of the protein markers Sox17, Cerberus, Hesx1, LeftyA, Otx1 orOtx2.

A forty-sixth aspect of the invention is a pharmaceutical compositioncomprising an effective amount of the population of the first,thirteenth, thirty-six and thirty-eighth aspects of the invention and acarrier.

A forty-seventh aspect of the invention is a substantially purifiedcomposition comprising one or more undifferentiated cells wherein thecells express a pancreatic progenitor cell marker protein. In apreferred embodiment the cells are embryonic stem cells. In anotherpreferred embodiment the cells are adult stem cells. In yet anotherpreferred embodiment the cells are hematopoietic stem cells and in stillanother preferred embodiment the cells are mesenchymal stem cells.

A forty-eighth aspect of the invention is the composition of theforty-seventh aspect which is transplanted into a subject. In apreferred embodiment the subject is a human subject.

A forty-ninth aspect of the invention is an in vivo method for inducingdifferentiation of resident pancreatic cells into islet cells comprisinga) introducing factors into the pancreas of a subject; and b) allowingthe introduced factors to prime the resident pancreatic cells such thatthe cells are induced to differentiate into islet progenitor cellsand/or islet cells. In a preferred embodiment the islet cells are alpha,beta, delta or phi cells.

A fiftieth aspect of the invention is an in vivo method for promotingthe generation of islet cells in a subject comprising a) transplantingamnion-derived cells into the pancreas of the subject; (b) introducingfactors into the pancreas of the subject; and c) allowing the introducedfactors to promote generation of islet progenitor cells or islet cellsfrom the transplanted amnion-derived cells. In a preferred embodimentthe amnion-derived cells are undifferentiated amnion-derived cells orpartially differentiated amnion-derived cells. In another embodiment thecells are transplanted subcutaneously, into liver, mammary gland, kidneycapsule, spleen or any other site in which the cells are able toengraft.

A fifty-first aspect of the invention is an in vivo method for promotingthe differentiation of amnion-derived cells into pancreatic cellscomprising (a) co-culturing the amnion-derived cells withdifferentiating embryonic pancreatic or non-pancreatic tissue; and (b)transplanting the co-cultures into the pancreas of a subject. In apreferred embodiment the non-pancreatic tissue is selected from thegroup consisting of epithelium, mesenchyme, islets, ducts, and exocrinetissue. In another preferred embodiment the amnion-derived cells areundifferentiated amnion-derived cells or partially differentiatedamnion-derived cells. In another embodiment the cells are transplantedsubcutaneously, into liver, mammary gland, kidney capsule, spleen or anyother site in which the cells are able to engraft.

A fifty-second aspect of the invention is an in vivo method forpromoting the differentiation of amnion-derived cells into pancreaticcells comprising (a) co-culturing the amnion-derived cells withdifferentiating or pre-differentiating non-embryonic heterologous orautologous tissue; and (b) transplanting the co-cultures into thepancreas of a subject. In a preferred embodiment the amnion-derivedcells are undifferentiated amnion-derived cells or partiallydifferentiated amnion-derived cells. In another embodiment the cells aretransplanted subcutaneously, into liver, mammary gland, kidney capsule,spleen or any other site in which the cells are able to engraft.

A fifty-third aspect of the invention is an in vivo method for promotingthe differentiation of amnion-derived cells into pancreatic cellscomprising (a) introducing factors to the amnion-derived cells in vitro;and (b) subsequently transplanting the amnion-derived cells into thepancreas of a subject. In a preferred embodiment the amnion-derivedcells are undifferentiated amnion-derived cells or partiallydifferentiated amnion-derived cells. In another embodiment the cells aretransplanted subcutaneously, into liver, mammary gland, kidney capsule,spleen or any other site in which the cells are able to engraft.

A fifty-fourth aspect of the invention is a cell culture systemcomprising a cell culture medium comprising a SHh antagonist and thepopulation of the first aspect or the thirty-sixth aspect of theinvention. In a preferred embodiment the cell culture system furthercomprises one or more mammalian embryonic islet progenitor cells. Inanother preferred embodiment the SHh antagonist is cyclopamine orjervine. In a specific preferred embodiment the cyclopamine is at aconcentration of 10 μM. In another preferred embodiment the cell culturesystem further comprises a solid surface. In a specific embodiment thesolid is extracellular matrix and in another specific embodiment theextracellular matrix is composed of one or more of the substancesselected from the group consisting of Matrigel, fibronectin,superfibronectin, laminin, collagen, heparin sulfate proteoglycan andnaturally occurring acellular biological substances. In anotherembodiment the solid surface forms a scaffold and in a specificembodiment the scaffold is a fiber, gel, fabric, sponge-like sheet orcomplex three-dimensional form containing pores and channels.

A fifty-fifth aspect of the invention is a cell culture systemcomprising a cell culture medium comprising a TAT fusion peptide and thepopulation of the first aspect or the thirty-sixth aspect of theinvention. In preferred embodiments the TAT fusion protein is TAT-PDX1,TAT-Hblx9, TAT-Ngn3, TAT-p48, or TAT-Foxa2. In a preferred embodiment,the cell culture system further comprises a SHh antagonist. In anotherpreferred embodiment the cell culture system further comprises one ormore mammalian embryonic islet progenitor cells. In another preferredembodiment the SHh antagonist is cyclopamine or jervine. In a specificpreferred embodiment the cyclopamine is at a concentration of 10 μM. Inanother preferred embodiment the cell culture system further comprises asolid surface. In a specific embodiment the solid is extracellularmatrix and in another specific embodiment the extracellular matrix iscomposed of one or more of the substances selected from the groupconsisting of Matrigel, fibronectin, superfibronectin, laminin,collagen, heparin sulfate proteoglycan and naturally occurring acellularbiological substances. In another embodiment the solid surface forms ascaffold and in a specific embodiment the scaffold is a fiber, gel,fabric, sponge-like sheet or complex three-dimensional form containingpores and channels.

A fifty-sixth aspect of the invention is a method for obtaining apancreatic progenitor cell comprising culturing an undifferentiated cellin the culture system of the fifty-fifth aspect of the invention.

A fifty-seventh aspect of the invention is a composition comprising adonor cell comprising a nucleus isolated from the amnion-derived cell ofthe first aspect of the invention. In a preferred embodiment the amnionderived cell is an expanded amnion-derived cell of the twenty-thirdaspect of the invention. In another preferred embodiment amnion-derivedcell is a pancreatic progenitor cell of aspect thirty-six of theinvention. In another preferred embodiment the amnion-derived cell is analpha, beta, delta or phi cell. In another preferred embodiment therecipient cell is a mammalian cell and in a specific embodiment themammalian cell is selected from the group consisting of germ cells,oocytes and sperm.

DEFINITIONS

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunction is retained by the polypeptide. NH2 refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refers to thefree carboxy group present at the carboxy terminus of a polypeptide.

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

As defined herein, a “gene” is the segment of DNA involved in producinga polypeptide chain; it includes regions preceding and following thecoding region, as well as intervening sequences (introns) betweenindividual coding segments (exons).

As used herein, the term “protein marker” means any protein moleculecharacteristic of the plasma membrane of a cell or in some cases of aspecific cell type.

As used herein, “enriched” means to selectively concentrate or toincrease the amount of one or more materials by elimination of theunwanted materials or selection and separation of desirable materialsfrom a mixture (i.e. separate cells with specific cell markers from aheterogenous cell population in which not all cells in the populationexpress the marker).

As used herein, the term “substantially purified” means a population ofcells substantially homogeneous for a particular marker or combinationof markers. By substantially homogeneous is meant at least 90%, andpreferably 95% homogeneous for a particular marker or combination ofmarkers.

As used herein, the term “monoclonal antibody library” means acollection of at least one monoclonal antibody useful for identifyingunique amnion-derived cells protein markers or generating substantiallypurified populations of amnion-derived cells. As defined herein,“specific for” means that the antibody(ies) specifically bind toamnion-derived cells, but not embryonic stem cells, mesenchymal stemcells or adult-derived stem cells.

The term “placenta” as used herein means both preterm and term placenta.

As used herein, the term “totipotent cells” shall have the followingmeaning. In mammals, totipotent cells have the potential to become anycell type in the adult body; any cell type(s) of the extraembryonicmembranes (e.g., placenta). Totipotent cells are the fertilized egg andapproximately the first 4 cells produced by its cleavage.

As used herein, the term “pluripotent stem cells” shall have thefollowing meaning. Pluripotent stem cells are true stem cells with thepotential to make any differentiated cell in the body, but cannotcontribute to making the components of the extraembryonic membraneswhich are derived from the trophoblast. The amnion develops from theepiblast, not the trophoblast. Three types of pluripotent stem cellshave been confirmed to date: Embryonic Stem (ES) Cells (may also betotipotent in primates), Embryonic Germ (EG) Cells, and EmbryonicCarcinoma (EC) Cells. These EC cells can be isolated fromteratocarcinomas, a tumor that occasionally occurs in the gonad of afetus. Unlike the other two, they are usually aneuploid.

As used herein, the term “multipotent stem cells” are true stem cellsbut can only differentiate into a limited number of types. For example,the bone marrow contains multipotent stem cells that give rise to allthe cells of the blood but may not be able to differentiate into othercells types.

“Amnion-derived cells” are a population of cells that are derived fromthe amnion of the placenta. Amnion-derived cells grow without feederlayers, do not express the protein telomerase and are non-tumorigenic.Amnion-derived cells do not express the hematopoietic stem cell markerCD34 protein. The absence of CD34 positive cells in this populationindicates the isolates are not contaminated with hematopoietic stemcells such as umbilical cord blood or embryonic fibroblasts. Virtually100% of the cells react with antibodies to low molecular weightcytokeratins, confirming their epithelial nature. Freshly isolatedamnion-derived cells will not react with antibodies to thestem/progenitor cell markers c-kit and Thy-1. Several procedures used toobtain cells from full term or pre-term placenta are known in the art(see, for example, US 2004/0110287; Anker et al., 2005, Stem Cells22:1338-1345; Ramkumar et al., 1995, Am. J. Ob. Gyn. 172:493-500).However, the methods used herein provide improved compositions andpopulations of cells.

The term “composition of placental-derived cells” as used hereinincludes the cells and compositions described in this application and inUS2003/0235563, US2004/0161419, US2005/0124003, U.S. ProvisionalApplication Nos. 60/666,949, 60/699,257, 60/742,067 and U.S. applicationSer. No. 11/333,849, the contents of which are incorporated herein byreference in their entirety.

By the term “animal-free” when referring to compositions, growthconditions, culture media, etc. described herein, is meant that noanimal-derived materials, such as animal-derived serum, other than humanmaterials, such as native or recombinantly produced human proteins, areused in the preparation, growth, culturing, expansion, or formulation ofthe composition or process.

By the term “expanded”, in reference to amnion-derived cellcompositions, means that the amnion-derived cell population constitutesa significantly higher concentration of multipotent cells than isobtained using previous methods. The level of multipotent cells per gramof amniotic tissue in expanded compositions is at least 50 and up to 150fold higher than the number of cells in the primary culture after 5passages, as compared to about a 20 fold increase in such cells usingprevious methods. Accordingly, an “expanded” population has at least a 2fold, and up to a 10 fold, improvement in cell numbers per gram ofamniotic tissue over previous methods. The term “expanded” is meant tocover only those situations in which a person has intervened to elevatethe proportion of the amnion-derived cells. As used herein “passage” or“passaging” refers to subculturing of cells. For example, cells isolatedfrom the amnion are referred to as primary cells. Such cells areexpanded in culture by being grown in the growth medium describedherein. When such primary cells are subcultured, each round ofsubculturing is referred to as a passage. As used herein, “primaryculture” means the freshly isolated amnion-derived cell population.

As used herein a “conditioned medium” is a medium in which a specificcell or population of cells has been cultured, and then removed. Whencells are cultured in a medium, they may secrete cellular factors thatcan provide support to or affect the behavior of other cells. Suchfactors include, but are not limited to hormones, cytokines,extracellular matrix (ECM), proteins, vesicles, antibodies, andgranules. The medium containing the cellular factors is the conditionedmedium. Examples of methods of preparing conditioned media are describedin U.S. Pat. No. 6,372,494 which is incorporated by reference in itsentirety herein. As used herein, conditioned medium also refers tocomponents, such as proteins, that are recovered and/or purified fromconditioned medium or from amnion-derived cells.

The term “lysate” as used herein refers to the composition obtained whenthe amnion-derived cell are lysed and the cellular debris (e.g.,cellular membranes) is removed. This may be achieved by mechanicalmeans, by freezing and thawing, by use of detergents, such as EDTA, orby enzymatic digestion using, for example, hyaluronidase, dispase,proteases, and nucleases.

As used herein, the term “substrate” means a defined coating on asurface that cells attach to, grown on, and/or migrate on. As usedherein, the term “matrix” means a substance that cells grow in or onthat may or may not be defined in its components. The matrix includesboth biological and non-biological substances. As used herein, the term“scaffold” means a three-dimensional (3D) structure (substrate and/ormatrix) that cells grow in or on. It may be composed of biologicalcomponents, synthetic components or a combination of both. Further, itmay be naturally constructed by cells or artificially constructed. Inaddition, the scaffold may contain components that have biologicalactivity under appropriate conditions.

The term “transplantation” refers to the administration of a compositioneither in an undifferentiated, partially differentiated, or fullydifferentiated form into a human or other animal.

As used herein, the term “pharmaceutically acceptable” means that thecomponents, in addition to the therapeutic agent, comprising theformulation, are suitable for administration to the patient beingtreated in accordance with the present invention.

The term “liver disease” as used herein includes but is not limited tocirrhosis of the liver, metabolic diseases of the liver, such as alpha1-antitrypsin deficiency and ornithine transcarbamylase (OTC),alcohol-induced hepatitis, chronic hepatitis, primary sclerosingcholangitis, alpha 1-antitrypsin deficiency and liver cancer. As usedherein, the term “pancreatic disease” may include but is not limited topancreatic cancer, insulin-deficiency disorder such as Insulin-dependent(Type 1) diabetes mellitus (IDDM) and Non-insulin-dependent (Type 2)diabetes mellitus (NIDDM), hepatitis C infection, exocrine and endocrinepancreatic diseases. As used herein, the term “neurological disease”refers to a disease or condition associated with any defects in theentire integrated system of nervous tissue in the body: the cerebralcortex, cerebellum, thalamus, hypothalamus, midbrain, pons, medulla,brainstem, spinal cord, basal ganglia and peripheral nervous system. Asused herein, the term “vascular disease” refers to a disease of thehuman vascular system. As used herein, the term “cardiac disease” or“cardiac dysfunction” refers to diseases that result from any impairmentin the heart's pumping function. The term “cardiomyopathy” refers to anydisease or dysfunction of the myocardium (heart muscle) in which theheart is abnormally enlarged, thickened and/or stiffened.

As used herein, the term “hepatocytes” refers to cells that havecharacteristics of epithelial cells obtained from liver. As used herein,the term “pancreatic cell” is used to refer to cells that produceglucagon, insulin, somatostatin, and/or pancreatic polypeptide (PP).Preferred pancreatic cells are positive for pancreatic cell-specificmarkers, such as homeobox transcription factor Nkx-2.2, glucagon, pairedbox gene 6 (Pax6), pancreatic duodenal homeobox 1 (PDX1), and insulin.As used herein, the term “vascular endothelial cell” refers to anendothelial cell that exhibits essential physiological functionscharacteristic of vascular endothelial cells including modulation ofvasoreactivity and provision of a semi-permeable barrier to plasma fluidand protein. As used herein, the term “cardiomyocyte” refers to acardiac muscle cell that may spontaneously beat or may exhibit calciumtransients (flux in intracellular calcium concentrations measurable bycalcium imaging). As used herein, the term “neural cells” refer to cellsthat exhibit essential functions of neurons, and glial cells (astrocytesand oligodendrocytes).

As used herein, the term “tissue” refers to an aggregation of similarlyspecialized cells united in the performance of a particular function.

As used herein, the term “therapeutic protein” includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors.

As used herein, “pancreas” refers generally to a large, elongated,racemose gland situated transversely behind the stomach, between thespleen and duodenum. The pancreatic exocrine function, e.g., externalsecretion, provides a source of digestive enzymes. These cellssynthesize and secrete digestive enzymes such as trypsinogen,chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease,triacylglycerol lipase, phospholipase A2 elastase, and amylase. Theendocrine portion of the pancreas contains the islets of Langerhans. Theislets of Langerhans appear as rounded spheroids of cells embeddedwithin the exocrine pancreas. Four different types of cells—alpha, beta,delta, and phi—have been identified in the islets. The alpha cellsconstitute about 20% of the cells found in pancreatic islets and producethe hormone glucagon. Glucagon acts on several tissues to make energyavailable in the intervals between feeding. In the liver, glucagoncauses breakdown of glycogen and promotes gluconeogenesis from aminoacid precursors. The delta cells produce somatostatin which acts in thepancreas to inhibit glucagon release and to decrease pancreatic exocrinesecretion. The hormone pancreatic polypeptide (PP) is produced in thephi cells. This hormone inhibits pancreatic exocrine secretion ofbicarbonate and enzymes, causes relaxation of the gallbladder, anddecreases bile secretion. The most abundant cell in the islets,constituting 60-80% of the cells, is the beta cell, which producesinsulin. Insulin is known to cause the storage of excess nutrientsarising during and shortly after feeding. The major target organs forinsulin are the liver, muscle, and fat-organs specialized for storage ofenergy. The term “pancreatic duct” as used herein includes the accessorypancreatic duct, dorsal pancreatic duct, main pancreatic duct andventral pancreatic duct, interlobular pancreatic duct, and interlobularpancreatic duct.

As used herein, the term “clustered amnion-derived cell compositions”refers to amnion-derived cell compositions wherein at least 50% and upto about 95% of the cells form clusters.

“Pancreatic progenitor cell” as defined herein is a cell which candifferentiate into a cell of pancreatic lineage, e.g., a cell which canproduce a hormone or enzyme normally produced by a pancreatic cell. Forinstance, a pancreatic progenitor cell may be caused to differentiate,at least partially, into alpha, beta, delta, or phi islet cells, or acell of exocrine fate. In accordance with the method of the invention,the pancreatic progenitor cells of the invention can be cultured priorto administration to a subject under conditions which promote cellproliferation and/or differentiation. These conditions include but arenot limited to culturing the cells to allow proliferation in vitro atwhich time the cells may form pseudo islet-like spheroids and secreteinsulin, glucagon, and somatostatin. The term “islet-like cell” as usedherein means having some but not necessarily all of the characteristicsof one of the cell types (α, β, γ or δ) present in a mature pancreaticislet. The islet-like cell will express only one of the followingpancreatic endocrine cell hormones: Insulin, glucagon, Somatostatin,Pancreatic Polypeptide. The term “islet-like structures” as definedherein are structures containing islet-like cells. Islet-like structuresrefers to the spheroids of cells derived from the methods of theinvention which take on both the appearance of pancreatic alpha, beta,delta or phi cells, as well as their function. Their coordinatedfunction includes the ability to respond to glucose.

As used herein, the term “spheroid” or “spheroids” means multicellularclusters in suspension cultures. As used herein the term “bud” or “buds”means the segregation of a subset of cells in a spheroid into a group onthe surface of the spheroid.

As used herein “germ cells” means embryonic germ cells, adult germ cellsand the cells that they give rise to (i.e. oocyte and sperm).

As used herein, “cloning” refers to producing an animal that developsfrom the combination of an oocyte and the genetic information containwithin the nucleus or the nucleic acid sequence of another animal, theanimal being cloned. The resulting oocyte having the donor genome isreferred to herein as a “nuclear transfer cell.” The cloned animal hassubstantially the same or identical genetic information as that of theanimal being cloned. “Cloning” may also refer to cloning a cell, whichincludes producing an oocyte containing genetic information from thenucleus or the nucleic acid sequence of another animal. Again, theresulting oocyte having the donor genome is referred to herein as a“nuclear transfer cell.”

The term “transplantation” as used herein refers to the administrationof a composition comprising cells that are either in anundifferentiated, partially differentiated, or fully differentiated forminto a human or other animal.

“Treatment” as used herein covers any treatment of a disease orcondition of a mammal, particularly a human, and includes: (a)preventing the disease or condition from occurring in a subject whichmay be predisposed to the disease or condition but has not yet beendiagnosed as having it; (b) inhibiting the disease or condition, i.e.,arresting its development; or (c) relieving the disease or condition,i.e., causing regression of the disease or condition. The population ofsubjects treated by the methods of the invention includes subjectssuffering from the undesirable condition or disease, as well as subjectsat risk for development of the condition or disease.

A “wound” is any disruption, from whatever cause, of normal anatomyincluding but not limited to traumatic injuries such as mechanical,thermal, and incisional injuries; elective injuries such as surgery andresultant incisional hernias; acute wounds, chronic wounds, infectedwounds, and sterile wounds, as well as wounds associated with diseasestates (i.e. ulcers caused by diabetic neuropathy). A wound is dynamicand the process of healing is a continuum requiring a series ofintegrated and interrelated cellular processes that begin at the time ofwounding and proceed beyond initial wound closure through arrival at astable scar. These cellular processes are mediated or modulated byhumoral substances including but not limited to cytokines, lymphokines,growth factors, and hormones. In accordance with the subject invention,“wound healing” refers to improving, by some form of intervention, thenatural cellular processes and humoral substances such that healing isfaster, and/or the resulting healed area has less scaring and/or thewounded area possesses tissue tensile strength that is closer to that ofuninjured tissue.

Definitions of additional terms are set forth in the table ofabbreviations below.

TABLE 1 Abbreviation Description Abbreviation Description A1AT Alpha-1Antitrypsin IE Islet equivalent CD34 Clustered Differentiation LeftyAEndometrial bleeding Antigen 34 associated factor preprotein c-Kit StemCell Factor Receptor MBP Myelin basic protein C/EBPα CCAAT/enhancerbinding Nkx 2.2 NK2 transcription factor protein-alpha related, locus 2CNP Natriuretic Peptide C IE Islet equivalent CYP Cytochrome Oct-4Octamer binding protein 3/4 ELISA Enzyme-Linked Pax Paired homeobox geneImmunosorbent Assay EROD Ethoxyresorufin-o- PCR Polymerase chainreaction deethylase EG Embryonic Germ PDX1 Pancreatic duodenal homeoboxprotein-1 ES Embryonic Stem PP Pancreatic Polypeptide FCS Fetal CalfSerum Rex-1 Reduced expression-1 FGF Fibroblast growth factor RIA RadioImmuno-Assay FACS Fluorescence Activated Cell Rt-PCR Quantitativereal-time Sorting polymerase chain reaction Foxa2 Forkhead box proteinA2; RT-PCR Reverse Transcription Hepatocyte nuclear factor 3- polymerasechain reaction beta GABA Gamma-amino butyric acid SHh Sonic Hedgehog GADGlutamic acid Sox-2 SRY-related HMG-box 2 decarboxylase HB9 HomeoboxProtein-HB9 SSEA Stage Specific Embryonic Antigen HNF4α Hepatocytenuclear factor- TDGF-1 Teratocarcinoma-derived 4α growth factor 1 HNF6Hepatocyte nuclear factor-6 Thy-1 Thymus cell antigen-1; CD90 ICCImmunocytochemistry TRA 1-60 Tumor related antigen-1-60 IHCImmunohistochemistry TRA 1-81 Tumor related antigen-1-81 ICM Inner CellMass UGT1A1 Uridine diphosphate glucuronosyltransferase

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A schematic representation of human embryological development.

FIG. 2—The application of conditioned media overcomes the inhibition ofwound healing caused by bacteria and shifts the healing trajectory incontaminated wounds to that of near normal healing.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Production of Amnion-Derived Cell Compositions

In accordance with the invention, amnion-derived cell compositions areprepared using the steps of a) recovery of the amnion from the placenta,b) dissociation of the cells from the amniotic membrane, c) culturing ofthe cells in a basal medium with the addition of a naturally derived orrecombinantly produced human protein; and optionally d) furtherproliferation of the cells using additional additives and/or growthfactors.

Recovery of the amnion—The first step in obtaining the amnion-derivedcell compositions of the invention is recovery of the cells from aplacenta. In general, the placenta is processed as soon as possibleafter delivery. In preferred embodiments, the placenta is processedwithin four hours of delivery. If the placenta is refrigerated, or theamnion is stripped and refrigerated, the recovery may be done in up to36 hours. The placenta used may be full term or pre-term placenta.Several procedures used to obtain cells from full term or pre-termplacenta are known in the art (see, for example, US 2004/0110287; Ankeret al., 2005, Stem Cells 22:1338-1345; Ramkumar et al., 1995, Am. J. Ob.Gyn. 172:493-500). However, the methods used herein provide improvedcompositions and populations of cells.

Under sterile conditions the amnion is stripped from the chorionmanually, and placed in Hanks Balanced Salt Solution (HBSS) with noadditives. This is preferably done at room temperature. The membranesare washed at least three times in HBSS, and washed further if necessaryto remove remaining blood clots. Any tissue still heavily contaminatedwith blood is cut away and discarded.

Dissociation of amniotic membrane cells—The membranes are incubated witha dissociation reagent. This is done at least once and as many as tentimes. In some embodiments, the dissociation reagent is a protease. Inpreferred embodiments, the protease is Protease XXIII (Sigma; 1 mg/ml).In other embodiments, the dissociation reagents include, but are notlimited to: trypsin±EDTA, papain, elastase, hyaluronidase, collagenasetype I, II, III, and IV, DNase, Ca+2 and Mg+2-free PBS, EDTA, EGTA,dispase, collagenase-dispase, Tryple (Gibco), collagenase, and dispase.

Characterizing, Identifying Isolating and Creating SubstantiallyPurified Populations of Amnion-Derived Cells.

Using commercially available antibodies to known stem cell markers,freshly isolated amnion-derived cells have been extensivelycharacterized. As set forth in Example 7, freshly isolatedamnion-derived cells are substantially purified with respect to CD90 andCD117. In addition, such populations are essentially negative forprotein expression of CD34, CD44, CD45, CD140b, CD105; essentiallypositive for protein expression of CD9 and CD29; between about 70-95%positive for protein expression of SSEA4, CD10, CD166 and CD227; betweenabout 60-95% positive for protein expression of HLA-G, EGFR and CD26;and between about 10-50% positive for protein expression of CD71.

In alternative embodiments substantially purified amnion-derived cellpopulations can be created using antibodies against protein markersexpressed (positive selection) or not expressed (negative selection) onthe cell surface of the amnion-derived cells. For instance, Example 8below demonstrates how antibodies can be used to create substantiallypurified populations. These antibodies may be used to identify,characterize, isolate or create such substantially purified populationsof amnion-derived cells expressing those protein markers using a varietyof methods. Such procedures may involve a positive selection, such aspassage of sample cells over a column containing anti-protein markerantibodies or by binding of cells to magnetic bead-conjugated antibodiesto the protein markers or by panning on plates coated with proteinmarker antibodies and collecting the bound cells. Alternatively, asingle-cell suspension may be exposed to one or more fluorescent-labeledantibodies that immuno-specifically bind to amnion-derived cell proteinmarkers. Following incubation with the appropriate antibody orantibodies, the amnion-derived cells are rinsed in buffer to remove anyunbound antibody. Amnion-derived cells expressing the protein marker(s)can then be sorted by fluorescence-activated cell sorting (FACS) using,for example, a Becton Dickinson FACStar flow cytometer. To createsubstantially purified populations of amnion-derived cells expressing adesired protein marker(s), the cells may be subjected to multiple roundsof FACS sorting.

In addition, protein markers that are not expressed on the surface ofamnion-derived cells may also be used to identify, isolate or createpopulations of amnion-derived cells not expressing those markers. Suchprocedures may involve a negative selection method, such as passage ofsample cells over a column containing anti-protein marker antibodies orby binding of cells to magnetic bead-conjugated antibodies to theprotein markers or by panning on plates coated with protein markerantibodies and collecting the unbound cells. Alternatively, asingle-cell suspension may be exposed to one or more fluorescent-labeledantibodies that immuno-specifically bind to the protein markers.Following incubation with the appropriate antibody or antibodies, thecells are rinsed in buffer to remove any unbound antibody. Cellsexpressing the protein marker(s) can then be sorted byfluorescence-activated cell sorting (FACS) using, for example, a BectonDickinson FACStar flow cytometer and these cells can be removed.Remaining cells that do not bind to the antibodies can then becollected. To create substantially purified populations ofamnion-derived cells that do not express a desired protein marker(s),the cells may be subjected to multiple rounds of FACS sorting asdescribed above.

The present invention further contemplates novel antibodies toamnion-derived cells or to amnion-derived cell protein markers describedherein. The antibodies are useful for detection of the amnion-derivedcell protein markers in, for example, diagnostic applications. Forpreparation of monoclonal antibodies directed toward amnion-derivedcells or to amnion-derived cell protein markers, any technique whichprovides for the production of antibody molecules by continuous celllines in culture may be used. For example, various protocols for theproduction of monoclonal antibodies can be found in Monoclonal AntibodyProtocols, Margaret E. Shelling, Editor, Humana Press; 2nd edition (Mar.15, 2002). In addition, the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in “Monoclonal Antibodies andCancer Therapy,” Alan R. Liss, Inc. pp. 77-96) and the like are withinthe scope of the present invention.

The monoclonal antibodies may be human monoclonal antibodies or chimerichuman-mouse (or other species) trionoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art (eg, Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozboret al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth.Enzymol. 92:3-16). Chimeric antibody molecules may be preparedcontaining a mouse antigen-binding domain with human constant regions(Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda etal., 1985, Nature 314:452).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of the amnion-derived cells or to theamnion-derived cell protein markers described herein. For the productionof antibody, various host animals can be immunized by injection withamnion-derived cells or to amnion-derived cell protein markers, or afragment or derivative thereof, including but not limited to rabbits,mice and rats. Various adjuvants may be used to increase theimmunological response, depending on the host species, and including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

A molecular clone of an antibody to selected amnion-derived cells or toamnion-derived cell protein marker epitope(s) can be prepared by knowntechniques. Recombinant DNA methodology (Sambrook et al, 2001,“Molecular Cloning: A Laboratory Manual”; Ausubel, ed., 1994, “CurrentProtocols in Molecular Biology” Volumes I-III) may be used to constructnucleic acid sequences which encode a monoclonal antibody molecule, orantigen binding region thereof.

The present invention provides for antibody molecules as well asfragments of such antibody molecules. Antibody fragments which containthe idiotype of the molecule can be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab′)2fragment which can be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which can be generated by reducing thedisulfide bridges of the F(ab′)2 fragment, and the Fab fragments whichcan be generated by treating the antibody molecule with papain and areducing agent. Another antibody fragment is the single chain Fv (scFv)which is a truncated Fab having only the V region of a heavy chainlinked by a stretch of synthetic peptide to a V region of a light chain.See, for example, U.S. Pat. Nos. 5,565,332; 5,733,743; 5,837,242;5,858,657; and 5,871,907 assigned to Cambridge Antibody TechnologyLimited incorporated by reference herein. Antibody molecules may bepurified by known techniques, e.g., immunoabsorption or immunoaffinitychromatography, chromatographic methods such as HPLC (high performanceliquid chromatography), or a combination thereof.

Expanded Populations of Amnion-Derived Cells

As described herein, Applicants have discovered a novel method forisolation and propagation of pluripotent, amnion-derived cells. Suchmethods result in amnion-derived cell compositions which are expandedfor pluripotent cells, thereby providing, for the first time, sufficientquantities of cells to enable therapeutic cell transplantation. Expandedamnion-derived cell compositions, which are made in accordance with thesubject invention, are compositions in which the level of multipotentcells per gram of amniotic tissue is at least 50 fold and up to 150 foldhigher after 5 passages, as compared to about 20 fold using previousmethods.

Additionally, the methods used for cell culture and proliferationprovide a means to culture the cells, as well as other pluripotentcells, including, but not limited to, embryonic stem cells, in ananimal-free system. Furthermore, the culture conditions describedprovide a cell that is less dependent on attachment to a culture surfacefor viability, thus allowing for propagation of the cells usingsuspension culture for efficient scale-up.

The expanded amnion-derived cell compositions described hereindemonstrate extensive proliferative potential, express certain genesknown to be expressed only in undifferentiated cells (i.e. Nanog andOct-4) and can differentiate into cell types that normally arise fromall three embryonic germ layers (endoderm, ectoderm and mesoderm). Thisdifferentiation potential suggests that these expanded amnion-derivedcells may be able to contribute to a variety of cell types. Theamnion-derived cell compositions described herein are also useful asfeeder layers for the growth of a variety of cell types, including butnot limited to embryonic stem cells (ES cells). Amnion-derived cells,including those described herein, also produce a wide variety ofcytokines and growth factors, thereby making both the cell compositions,conditioned medium derived from the cells, cell lysates thereform,extracellular matrices produced by the cells, and combinations thereofuseful to achieve rapid and effective wound healing, including scarlesshealing, and also useful in cosmetics, i.e. to achieve improvement inskin appearance.

Culturing of the amniotic cells—The cells are cultured in a basalmedium. Such medium includes, but is not limited to, Epilife (CascadeBiologicals), Opti-pro, VP-SFM, IMDM, Advanced DMEM, K/O DMEM, 293 SFMII (all made by Gibco; Invitrogen), HPGM, Pro 293S-CDM, Pro 293A-CDM,UltraMDCK, UltraCulture (all made by Cambrex), Stemline I and StemlineII (both made by Sigma-Aldrich), DMEM, DMEM/F-12, Ham's F12, M199, andother comparable basal media. Such media should either contain humanprotein or be supplemented with human protein. As used herein a “humanprotein” is one that is produced naturally or one that is produced usingrecombinant technology. “Human protein” also is meant to include a humanfluid or derivative or preparation thereof, such as human serum oramniotic fluid, which contains human protein. In preferred embodiments,the basal media is Stemline I or II, UltraCulture, or Opti-pro, orcombinations thereof and the human protein is human albumin at aconcentration of at least 0.5% and up to 10%. In particular embodiments,the human albumin concentration is from about 0.5 to about 2%. The humanalbumin may come from a liquid or a dried (powder) form and includes,but is not limited to, recombinant human albumin, plasbumin andplasmanate.

In a most preferred embodiment, the cells are cultured using a systemthat is free of animal products to avoid xeno-contamination. In thisembodiment, the culture medium is Stemline I or II, Opti-pro, or DMEM,with human albumin (plasbumin) added up to concentrations of 10%.Alternatively, UltraCulture may be used, with substitution oftransferrin with human recombinant transferrin, and replacement of thebovine albumin (BSA) with human albumin at concentrations of up to 10%.The invention further contemplates the use of any of the above basalmedia wherein animal-derived proteins are replaced with recombinanthuman proteins and animal-derived serum, such as BSA, is replaced withhuman albumin. In preferred embodiments, the media is serum-free inaddition to being animal-free.

Further, the culture conditions described herein result in the formationof three-dimensional clusters of cells called spheroids, a property thatmay enhance the likelihood of differentiation to, for example,pancreatic islet cells, neural lineages and cardiac cells. Suchcompositions are prepared as described above using a basal mediaselected from the group consisting of Opti-pro SFM, VP-SFM, Iscove'sMDM, HPGM, UltraMDCK, Stemline II and Stemline I, DMEM, and DMEM:F12with added human albumin, plasmanate or plasbumin at levels of up to10%.

In alternative embodiments, where the use of non-human serum is notprecluded, such as for in vitro uses, the culture medium may besupplemented with serum derived from mammals other than humans, inranges of up to 40%.

Additional proliferation—Optionally, other proliferation factors areused. In one embodiment, epidermal growth factor (EGF), at aconcentration of between 0-1 μg/ml is used. In a preferred embodiment,the EGF concentration is around 10 ng/ml. Alternative growth factorswhich may be used include, but are not limited to, TGFα or TGFβ (5ng/ml; range 0.1-100 ng/ml), activin A, cholera toxin (preferably at alevel of about 0.1 μg/ml; range 0-10 μg/ml), transferrin (5 μg/ml; range0.1-100 μg/ml), fibroblast growth factors (bFGF 40 ng/ml (range 0-200ng/ml), aFGF, FGF-4, FGF-8; (all in range 0-200 ng/ml), bone morphogenicproteins (i.e. BMP-4) or other growth factors known to enhance cellproliferation.

Passaging—Cells are initially plated at a density of25,000/cm²-1,000,000/cm², on tissue culture treated plates, preferablyat a density of about 130,000/cm². In one embodiment, the cells aregrown on extracellular matrix treated plates, such as collagen, laminin,fibronectin, or Matrigel. To create the expanded amnion-derived cellcompositions of the invention, the cells are passaged at least five (5)times as described below in Example 1. To create the spheroidalamnion-derived cell compositions of the invention, only one passage isrequired.

Growth of ES cells—The culture media described above may also be used toproduce expanded or spheroidal preparations of embryonic stem cells (EScells). In some embodiments, the culture medium is free of animalproducts. In preferred embodiments, the culture medium is free of animalproduct and is made without serum.

Large scale culture of amnion-derived cells—In further embodiments,large scale culture is used to produce the amnion-derived cellcompositions, conditioned media therefrom, and cells for the preparationof cell lysates. The literature describing large-scale mammalian cellculture has predominantly related to the culture of cells such asChinese hamster ovary (CHO) cells to produce therapeutic proteins(Moreira, J. L. et al. (1995) Biotechnol Prog, 11:575). In this case,the secretory product of the cells is the main product of interest.Technologies most often used for large-scale cell production have beenspinner flasks and roller bottles, although roller bottles are beingreplaced in some applications, especially adherent cell culture, byhollow-fibre culture bioreactors and microcarrier bioreactor systems(Martin, I., et al. (2004) Trends Biotechnol, 22:80). Hollow fibrebioreactors combine synthetic fibres with mammalian cells. The cells areseeded between the fibres and grow in a 3-dimensional tissue-likeformation. The fibres act as conduits for nutritional factors and oxygento reach the cells, and also provide an exit for toxins and cellularby-products which need to be removed from the proximity of the cells.One of the drawbacks of hollow-fibre technology is the difficulty inrecovery of the cells, although for some applications, such asextracorporeal hepatic assist devices, the cells remain in situ toperform their therapeutic purpose (Gerlach, J. C., (1997) Cell BiolToxicol, 13:349).

Large scale cell culture may be used to culture the amnion-derived cellsas a product for some therapeutic purposes, including both growth ofcells for transplantation, as well as for production of conditionedmedia. Hematopoietic cells for bone marrow transplant have been culturedin suspension (Cheshier, S. H., et al, (1999) Proc Natl Acad Sci USA,96:3120; Madlambayan, G. J., et al, (2001) J Hematother Stem Cell Res,10:481), hepatocytes for extracorporeal assist devices (Gerlach, J. C.,(1997) Cell Biol Toxicol, 13:349), keratinocytes for artificial skinapplications (Zacchi, V., et al, (1998) J Biomed Mater Res, 40:187;Pellegrini, G., et al, (1998) Med Biol Eng Comput, 36:778; Waymack, P.,et al, (2000) Burns, 26:609), and neural stem cells forneurodegenerative diseases (Kallos, M. S., et al. (2003) Med Biol EngComput, 41:271). If mammalian cells are anchorage-dependent and cannotbe cultured in suspension, microcarrier beads or microcarriers can beused as a large surface area to which the cells can attach and grow inthe suspension apparatus. The cell-covered microcarrier beads aremaintained in suspension in the apparati used for cell suspensioncultures, allowing for reductions in media usage and space requirements.One of the technical difficulties of microcarrier bead culture is theefficient removal of the cells from the beads themselves, withoutcompromising viability (Varani, J., et al. (1986) J Biol Stand, 14:331).The preferred method would be to culture amnion-derived cells insuspension.

The scalable production of amnion-derived cells may be accomplishedusing systems currently being developed for human embryonic stem (hES)cells. Most examples of scale-up for hES cells include partial orcomplete differentiation during the scale-up process, for instanceGerecht-Nir and coworkers (Gerecht-Nir, S., et al. (2004) BiotechnolBioeng, 86:493) report the scalability of ES cells as embryoid bodies.Other examples of differentiated scale-up for ES cells include cardiaccells (Zandstra, P. W., et al. (2003) Tissue Eng, 9:767) in whichembryoid bodies are formed and treated with retinoic acid, andES-derived hepatocyte scale-up in hollow fibre bioreactors (Gerlach, J.C., (1997) Cell Biol Toxicol, 13:349). Reports of scale-up ofundifferentiated human ES cells are scarce, although mouse ES cells canbe proliferated in hollow fibre bioreactors, with maintenance of theirstem cell surface characteristics.

In other embodiments, the cells are cultured in suspension cultureconditions including in suspension culture treated plates, and rollerbottles (in roller bottles density range 100,000/ml-5 million/ml;preferred 1 million/ml), or spinner flasks with or without attachment tomicrocarrier beads. Examples 2, 3 and 4 sets forth methods of largescale production that may be used in accordance with the invention.

Experiments are performed to determine the medium and supplements thatcan be used for optimal cell growth and expression of differentiatedfunction. The use of multiple spinner flasks permits the use of 2 or 3replicates of each condition per experiment.

Once cells are grown successfully in suspension, they are then culturedin sufficient quantities for transplantation. One skilled in the artwill recognize that the number of cells needed for transplantation willdepend upon the specific application. Cells are harvested as determinedin the first set of experiments above but instead of seeding intoT-flasks or spinner flasks they are put into Wave bags. These aresterile plastic bags (Wave, Inc.) into which cells and medium are added.The bag and its contents are placed on a rocker that gently agitates theentire bag. In addition, CO₂ and air can be added continuously to thebag to maintain adequate oxygen and pH control. The range of bag sizesis 1 liter to 1000 liters. In one embodiment, 1 liter bags and a minimumworking volume of 125 ml are used. As the cells grow, additional mediumcan be added until the 500 ml working volume is attained.

Amnion-derived cells are placed both into the Wave bags and into thenormal T-flasks and incubated at 37° C. at 5% CO₂ in air. Daily samplesof cells are withdrawn, in a class 100 biosafety cabinet, from eachflask and vessel, and the cells are stained with trypan blue and countedon a hemacytometer. A graph of total and viable cell counts per ml isplotted with time to ensure that amnion-derived cells divide and remainviable with time in culture.

Wave bags have two main advantages over alternative culture vessels.Firstly, they are disposable and therefore cleaning validation is notnecessary for each lot of cells. Secondly, because the rocking motioncreates a wave of liquid in the bags, the gas exchange in the liquid ismuch higher than if the cells were in a stationary flask or spinnerflask. As a result, the total attainable cell number is higher than ineither the T-flasks or spinner flasks.

At specific intervals, samples are analyzed using reverse transcriptaseand/or real time PCR for gene expression over time. The samples areexamined for amnion-derived cell-specific markers such as Oct-4, nanog,etc. In addition, samples are measured by FACS analysis for cell surfacemarkers of undifferentiated cells (SSEA-3 and 4, Tra-1-60 and Tra-1-81).

In addition, the ability of amnion-derived cells after suspensionculture and proliferation to undergo differentiation to all three germlineages (endoderm, mesoderm, and ectoderm) is analyzed. Suchcharacterization of differentiative capacity is assessed by performingdifferentiation assays and analysis of gene expression by reversetranscriptase and/or real-time PCR and by low volume FACS analysis (orIHC).

At designated time intervals, the cells are removed from the culturevessels and cultured in differentiation protocols to determine theirability to differentiate to the three germ lineages after proliferation.

Compositions—The compositions of the invention include substantiallypurified populations and pharmaceutical compositions of such. Thecompositions of the invention can be prepared in a variety of waysdepending on the intended use of the compositions. For example, acomposition useful in practicing the invention may be a liquidcomprising an agent of the invention, i.e. a substantially purifiedpopulation of amnion-derived cells, in solution, in suspension, or both(solution/suspension). The term “solution/suspension” refers to a liquidcomposition where a first portion of the active agent is present insolution and a second portion of the active agent is present inparticulate form, in suspension in a liquid matrix. A liquid compositionalso includes a gel. The liquid composition may be aqueous or in theform of an ointment, salve, cream, or the like.

An aqueous suspension or solution/suspension useful for practicing themethods of the invention may contain one or more polymers as suspendingagents. Useful polymers include water-soluble polymers such ascellulosic polymers and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. An aqueous suspension orsolution/suspension of the present invention is preferably viscous ormuco-adhesive, or even more preferably, both viscous and muco-adhesive.

Pharmaceutical Compositions—The present invention providespharmaceutical compositions of substantially purified populations ofamnion-derived cells, and a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the composition isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, and still othersare familiar to skilled artisans.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Treatment Kits—The invention also provides for an article of manufacturecomprising packaging material and a pharmaceutical composition of theinvention contained within the packaging material, wherein thepharmaceutical composition comprises a substantially purified populationof amnion-derived cells, and wherein the packaging material comprises alabel or package insert which indicates that the substantially purifiedpopulation of amnion-derived cells can be used for treating a variety ofdisorders including but not limited to diabetes, liver disease, neuraldisease, etc.

Amnion-Derived Cell Nuclei

In addition to amnion-derived cells themselves and products derivedtherefrom, another embodiment of the invention is the nuclei ofamnion-derived cells. Such nuclei may be obtained using methods known inthe art. These include removing the membranes from cells by eithermechanical disruption or chemical means such as treatment withhyaluronidase or performed by mechanically extracting the nucleus with apipet. The nuclei may then subsequently be transferred into somatic orgerm cells, by for example, intracytoplasmic injection or electrofusionusing methods known in the art as described in for example, US20030234430 or US 20040268422. As amnion-derived cells are derived fromextraembryonic tissue, specifically the amnion, they are non-somatic,non-fetal, and non-germ cells, thus unique among the donor cellstypically used in the art.

Once the nuclei are transferred into another cell, the resulting cellmay be used for any number of applications. One embodiment relates tomethods of therapeutic nuclear cloning or cloning an animal by combiningany enucleated cell with the nuclei from an amnion-derived cell. Thisembodiment encompasses the cloning of a variety of animals. Theseanimals include all mammals (e.g., human, canines, felines, mice, rats,livestock cattle, sheep, goats, camels, pigs, horses, llamas). The donoramnion-derived cell and the oocyte may or may not be from the sameanimal.

The genome of the donor amnion-derived cell can be the naturallyoccurring genome, for example, for the production of cloned animals, orthe genome can be genetically altered to comprise a transgenic sequence,for example, for the production of transgenic cloned animals.

The oocytes used in the present invention could be in any stage ofmeiotic cell division, including metaphase I, anaphase I, anaphase II,telophase I, telophase II, and preferably metaphase II. Oocytes inmetaphase II are considered to be in a resting state. The oocytes can bein the resting stage of metaphase II, and then be activated, usingmethods described herein. The stage that the oocyte is in can beidentified by visual inspection of the oocyte under a sufficientmagnification. Methods for identifying the stage of meiotic celldivision are known in the art.

Oocytes can be activated by physical (e.g. electrical stimulation, coldshock) and chemical means (e.g. ethanol, acid tyrode's solution,strontium chloride, calcium ionophore, puromycin, hyaluronidase andmedia lacking calcium and magnesium). Some of these methods activateoocytes by increasing intracellular calcium levels. Several methodsexist that allow for activation of the oocyte. In particular, a calciumionophore (e.g., ionomycin) is an agent that increases the permeabilityof the oocyte's membrane and allows calcium to enter into the oocyte.Such methods of activation are described in U.S. Pat. No. 5,496,720.Ethanol has a similar affect. Prior to or following enucleation, anoocyte in metaphase II can be activated with ethanol according to theethanol activation treatment as described in Presicce and Yang, Mol.Reprod. Dev., 37: 61-68 (1994); and Bordignon & Smith, Mol. Reprod.Dev., 49: 29-36 (1998). Exposure of calcium to the oocyte also occursthrough electrical stimulation. The electrical stimulation allowsincreasing levels of calcium to enter the oocyte. Other known methods ofactivation can be used with the present invention to activate theoocyte.

Oocytes can be obtained from a donor animal during that animal'sreproductive cycle. For example, oocytes can be aspirated from folliclesof ovaries at given times during the reproductive cycle (exogenoushormone-stimulated i.e. super-ovulation or ovarian hyperstimulation ornon-stimulated). Also at given times following ovulation, a significantpercentage of the oocytes, for example, are in telophase. Additionally,oocytes can be obtained and then induced to mature in vitro to anarrested metaphase II stage. Arrested metaphase II oocytes, produced invivo or in vitro, can then be induced in vitro to enter telophase. Thus,oocytes in telophase can readily be obtained for use in the presentinvention. Oocytes can also be recovered surgically by flushing theoocytes from the oviduct of a female donor. Methods for the collectionof oocytes are known in the art.

Preferably, the cell stage of the activated oocytes correlates to thestage of the cell cycle of the donor amnion-derived cell. Thiscorrelation between the meiotic stage of the oocyte and the mitoticstage of the donor cell is also referred to herein as “synchronization.”

The present invention utilizes an oocyte that is enucleated. Anenucleated oocyte is one that is devoid of the genome, or one that is“functionally enucleated.” A functionally enucleated oocyte contains agenome that is non-functional, e.g., cannot replicate or synthesize DNA.See, for example, Bordignon, V. and L. C. Smith, Molec. Reprod. Dev.,49: 29-36 (1998). Preferably, the genome of the oocyte is removed fromthe oocyte. A genome can be functionally enucleated from the oocyte byirradiation, by x-ray irradiation, by laser irradiation, by physicallyremoving the genome, or by chemical means. Methods of using irradiationare known to those in the art and are described, for example, inBradshaw et al., Molecul. Reprod. Dev., 41: 503-512 (1995). Methods ofchemically inactivating the DNA are known to those of skilled in the art(Fulka and Moore, Molecul. Reprod. Dev., 34: 427-430 (1993). Other knownmethods of enucleation can be used with the present invention toenucleate the oocyte.

To physically remove the genome of an oocyte, one can insert amicropipette or needle through the zona pellicuda of the oocyte toremove nuclear material from the oocyte. In one example, telophaseoocytes which have two polar bodies can be enucleated with amicropipette or needle by removing the second polar body in surroundingcytoplasm. Specifically, oocytes in telophase stage of meiosis can beenucleated at any point from the presence of a protrusion in the plasmamembrane from the second polar body up to the formation of the secondpolar body itself. Thus, as used herein, oocytes which demonstrate aprotrusion in the plasma membrane, usually with a spindle abutted to it,up to extrusion of a second polar body are considered to be oocytes intelophase. In another example, metaphase II stage oocytes can beenucleated by puncturing the zona pellucida with a micropipette,abutting the micropipette to the oocyte nucleus, withdrawing the nucleusand part of the oolemma (or oocytes membrane) into the pipette. Afterwithdrawal of the micropipette the oolemma pinches off to leave amembrane-intact enucleated oocyte. Oocytes can also be pre-treated withcytochalasin D to aid in this process.

The present invention includes enucleating the genome of an oocyte bytreating the oocyte with a compound that will induce the oocyte genome(e.g., nuclear chromatin) to segregate into the polar bodies duringmeiotic maturaton thereby leaving the oocyte devoid of a functionalgenome, and resulting in the formation of a recipient cytoplast for usein nuclear transfer procedures. Examples of agents that will effect suchdifferential segregation include agents that will disrupt cytoskeletaland metabolism (see, for example Andreau, J. M. and Timasheff, S, N.,Proc. Nat. Acad. Sci. 79: 6753 (1982), Obrig, T. G., et al, J. Biol.Chem. 246: 174 (1971), Duskin, D. and Mahoney, W. C., J. Biol. Chem.257: 3105 (1982), Scialli, A. R., et al, Teratogen, Carcinogen, Mutagen14: 23 (1994), Nishiyama, I. and Fujii, T., Exp. Cell Res. 198: 214(1992), Small, J. V., et al, J. Cell Sci. 89: 21 (1988), Lee, J. C., etal, Biochem. 19: 6209 (1980). The age of the oocyte and timing of theevents (i.e. enucleation, fusion and activation) are also very importantin successful nuclear transfer and are well known to those skilled inthe art.

Combination of the activated, enucleated oocyte and the genome from theamnion-derived cell can occur a variety of ways to form the nucleartransfer embryo. A genome of an amnion-derived cell can be injected intothe activated oocyte by employing a microinjector (i.e., micropipette orneedle). The nuclear genome of the amnion-derived cell is extractedusing a micropipette or needle. Once extracted, the amnion-derivedcell's nuclear genome can then be placed into the activated oocyte byinserting the micropipette, or needle, into the oocyte and releasing thenuclear genome of the amnion-derived cell. (McGrath, J. and D. Solter,Science, 226: 1317-1319 (1984)).

The present invention also includes combining the genome of anamnion-derived cell with an activated oocyte by fusion e.g.,electrofusion, viral fusion, liposomal fusion, biochemical reagentfusion (e.g., phytohemaglutinin (PHA) protein), or chemical fusion(e.g., polyethylene glycol (PEG) or ethanol). The amnion-derived cell,an amnion-derived cell karyoplast or the nucleus of the amnion-derivedcell can be deposited within the zona pellucida which contains theoocyte. The steps of fusing the cell, karyoplast or nucleus with theoocyte can then be performed by techniques known in the art. Thecombination of the genome of the amnion-derived cell with the activatedoocyte results in a nuclear transfer embryo.

A nuclear transfer cell of the present invention could then betransferred into a recipient non-human female animal and allowed todevelop or gestate into a cloned or transgenic animal. Conditionssuitable for gestation are those conditions that allow for the embryo todevelop and mature into a fetus, and eventually into a live animal. Suchconditions are known in the art. The nuclear transfer cell can bemaintained in a culture system until at least the first cleavage (2-cellstage) up to the blastocyst stage. Preferably the nuclear transfer cellsare transferred at the 2-cell or 4-cell stage. Various culture media fornuclear transfer cell development are known to those skilled in the art.

The present invention also relates to methods for generating transgenicanimals by combining an activated oocyte with a genetically engineeredgenome from an amnion-derived cell. Such a combination results in atransgenic nuclear transfer cell. A transgenic animal is an animal thathas been produced from a genome from a donor cell that has beengenetically altered, for example, to produce a particular protein (adesired protein), or that has been altered to knock-out a particulargene. Methods for introducing DNA constructs into the germ line of ananimal to make a transgenic animal are known in the art.

The present invention is also directed to “therapeutic cloning”, whichis the production of ES cells from a cloned embryo. Previously, Munsie,at al. reported the isolation of mouse ES cells from blastocysts derivedby somatic cell nuclear transfer (Current Biology 10: 989-992, 2000).Wakayama, et al. obtained mouse ES cells, which can be induced tovarious types of specific cells in vitro, from the cultures ofblastocysts derived by somatic cell nuclear transfer (Science, 292(5517); 740-743. 2001). The result of the research done by Wakayama, etal. demonstrates that ES cells can be isolated from nuclear transferembryos by somatic cell nuclear transfer. The nuclear transfer ES cellsof somatic cell origin are pluripotent and can differentiate into anyspecific cell types as ES cells derived from the normal zygote.

In the present invention, therapeutic cloning is accomplished by thenuclear transfer of an amnion-derived cell nucleus into an oocyte. Thenuclear transfer cells obtained are further differentiated into aspecific cell type needed by, for example, a patient suffering from adisease (i.e. diabetes, liver failure, etc.).

Therapeutic uses of Amnion-Derived Cells and Differentiated Cells

Because these compositions comprise much higher cell numbers peramniotic tissue than have previously been achieved, they allow fortherapeutic use in situations, such as transplantation, which requirelarge numbers of cells. These cells have been found to be multipotent,i.e. capable of differentiating into a variety of tissue types includingbut not limited to hematopoietic, liver, pancreatic, nervous, muscle andendothelial tissues. Such cells are particularly useful to restorefunction in diseased tissues via transplantation therapy or tissueengineering, and to study metabolism and toxicity of compounds in drugdiscovery efforts.

Cell transplantation strategies currently used in the clinic or inclinical trials have demonstrated promising results, e.g., 1) Pancreaticislets, isolated from cadaver tissue, are currently transplanted torestore proper insulin secretion and alleviate the need for insulininjections in Type I diabetic patients and 2) Hepatocytes isolated fromcadaver livers are transplanted to treat patients awaiting livertransplant and for treatment of metabolic disorders. However, the needfor clinical grade pancreatic islets and hepatocytes far exceeds thenumber of cells that can be isolated and transplanted from donor tissue.Expanded amnion-derived cell compositions described herein provide anabundant cell source that can be differentiated to these cell types.

Compositions comprising amnion-derived cell or cells differentiatedtherefrom may be administered to a subject to provide various cellularor tissue functions. As used herein “subject” may mean either a human ornon-human animal.

Such compositions may be formulated in any conventional manner using oneor more physiologically acceptable carriers optionally comprisingexcipients and auxiliaries. Proper formulation is dependent upon theroute of administration chosen. The compositions may be packaged withwritten instructions for use of the cells in tissue regeneration, orrestoring a therapeutically important metabolic function. Amnion-derivedcells may also be administered to the recipient in one or morephysiologically acceptable carriers. Carriers for these cells mayinclude but are not limited to solutions of phosphate buffered saline(PBS) or lactated Ringer's solution containing a mixture of salts inphysiologic concentrations.

One of skill in the art may readily determine the appropriateconcentration of cells for a particular purpose. A preferred dose is inthe range of about 0.25−1.0×10⁶ cells.

Amnion-derived cells or cells differentiated therefrom can beadministered by injection into a target site of a subject, preferablyvia a delivery device, such as a tube, e.g., catheter. In a preferredembodiment, the tube additionally contains a needle, e.g., a syringe,through which the cells can be introduced into the subject at a desiredlocation. Specific, non-limiting examples of administering cells tosubjects may also include administration by subcutaneous injection,intramuscular injection, or intravenous injection. If administration isintravenous, an injectable liquid suspension of cells can be preparedand administered by a continuous drip or as a bolus.

Cells may also be inserted into a delivery device, e.g., a syringe, indifferent forms. For example, the cells can be suspended in a solutioncontained in such a delivery device. As used herein, the term “solution”includes a pharmaceutically acceptable carrier or diluent in which thecells of the invention remain viable. Pharmaceutically acceptablecarriers and diluents include saline, aqueous buffer solutions, solventsand/or dispersion media. The use of such carriers and diluents is wellknown in the art. The solution is preferably sterile and fluid to theextent that easy syringability exists. Preferably, the solution isstable under the conditions of manufacture and storage and preservedagainst the contaminating action of microorganisms such as bacteria andfungi through the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention canbe prepared by incorporating amnion-derived cells or differentiatedcells as described herein, in a pharmaceutically acceptable carrier ordiluent and, as required, other ingredients enumerated above, followedby filter sterilization.

Undifferentiated, partially differentiated or fully differentiatedamnion-derived cells may be administered systemically (for exampleintravenously) or locally (for example directly into a myocardial defectunder echocardiogram guidance or by direct application undervisualization during surgery). For such injections, the cells may be inan injectable liquid suspension preparation or in a biocompatible mediumwhich is injectable in liquid form and becomes semi-solid at the site ofdamaged tissue. A conventional intra-cardiac syringe or a controllableendoscopic delivery device can be used so long as the needle lumen orbore is of sufficient diameter (e.g. 30 gauge or larger) that shearforces will not damage the cells being delivered.

Cells may be administered in a manner that permits them to graft to theintended tissue site and reconstitute or regenerate the functionallydeficient area. Undifferentiated, partially differentiated or fullydifferentiated amnion-derived cells can be used in therapy by directadministration, or as part of a bioassist device that provides temporaryor permanent organ function. In this respect, undifferentiated,partially differentiated or fully differentiated amnion-derived cellsmay be grown in a bioreactor to provide extracorporeal organ support fororgan relief, such as in the case of a liver assist device, to provide aplentiful source of cells for transplantation to restore organ function,or provide a source of conditioned medium that may be used to stimulatetissue regeneration. Liver assist devices utilizing primary porcinecells as well as primary human liver cells have been used successfully(Sauer, I. M., et al. Xenotransplantation (2003) 10:460-469; Irgang, M.et al. (2003) 28(2):141-154; Sauer, I. M. et al. (2002) Int. J. Art.Org. 25(10):1001-1006; Sauer, I. M. et al. (2002) J. Metabolic BrainDisease 17(4): 477-484, Sauer, I. M. et al. (2003) J. Hepatology39(4):649-653). Amnion-derived cell-derived hepatocytes may be utilizedin conjunction with this technology.

Alternatively, amnion-derived cells may be transplanted into therecipient where the cells will proliferate and differentiate to form newcells and tissues thereby providing the physiological processes normallyprovided by that tissue, or may produce factors that cause the migrationand/or differentiation of cells in the area of the transplant. Tissuesare an aggregation of similarly specialized cells united in theperformance of a particular function. Tissue is intended to encompassall types of biological tissue including both hard and soft tissue. Softtissue refers to tissues that connect, support, or surround otherstructures and organs of the body. Soft tissue includes muscles, tendons(bands of fiber that connect muscles to bones), fibrous tissues, fat,blood vessels, nerves, and synovial tissues (tissues around joints).Hard tissue includes connective tissue (e.g., hard forms such as osseoustissue or bone) as well as other muscular or skeletal tissue.

Support matrices into which the amnion-derived cells can be incorporatedor embedded include matrices which are recipient-compatible and whichdegrade into products which are not harmful to the recipient. Thesematrices provide support and protection for undifferentiated anddifferentiated amnion-derived cells in vivo and are, therefore, thepreferred form in which such cells are transplanted into the recipientsubjects.

Natural and/or synthetic biodegradable matrices are examples of suchmatrices. Natural biodegradable matrices include plasma clots, e.g.,derived from a mammal, collagen, fibronectin, and laminin matrices.Suitable synthetic material for a cell transplantation matrix must bebiocompatible to preclude migration and immunological complications, andshould be able to support extensive cell growth and differentiated cellfunction. It must also be resorbable, allowing for a completely naturaltissue replacement. The matrix should be configurable into a variety ofshapes and should have sufficient strength to prevent collapse uponimplantation. Recent studies indicate that the biodegradable polyesterpolymers made of polyglycolic acid fulfill all of these criteria(Vacanti, et al. J. Ped. Surg. 23:3-9 (1988); Cima, et al. Biotechnol.Bioeng. 38:145 (1991); Vacanti, et al. Plast. Reconstr. Surg. 88:753-9(1991)). Other synthetic biodegradable support matrices includesynthetic polymers such as polyanhydrides, polyorthoesters, andpolylactic acid. Further examples of synthetic polymers and methods ofincorporating or embedding cells into these matrices are also known inthe art. See e.g., U.S. Pat. Nos. 4,298,002 and 5,308,701.

Attachment of the cells to the polymer may be enhanced by coating thepolymers with compounds such as basement membrane components, agar,agarose, gelatin, gum arabic, collagens types I, II, III, IV and V,fibronectin, laminin, glycosaminoglycans, mixtures thereof, and othermaterials known to those skilled in the art of cell culture. Allpolymers for use in the matrix must meet the mechanical and biochemicalparameters necessary to provide adequate support for the cells withsubsequent growth and proliferation. The polymers can be characterizedwith respect to mechanical properties such as tensile strength using anInstron tester, for polymer molecular weight by gel permeationchromatography (GPC), glass transition temperature by differentialscanning calorimetry (DSC) and bond structure by infrared (IR)spectroscopy, with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies.

One of the advantages of a biodegradable polymeric matrix is thatangiogenic and other bioactive compounds can be incorporated directlyinto the support matrix so that they are slowly released as the supportmatrix degrades in vivo. As the cell-polymer structure is vascularizedand the structure degrades, amnion-derived cells may differentiateaccording to their inherent characteristics. Factors, includingnutrients, growth factors, inducers of differentiation orde-differentiation (i.e., causing differentiated cells to losecharacteristics of differentiation and acquire characteristics such asproliferation and more general function), products of secretion,immuno-modulators, inhibitors of inflammation, regression factors,biologically active compounds which enhance or allow ingrowth of thelymphatic network or nerve fibers, hyaluronic acid, and drugs, which areknown to those skilled in the art and commercially available withinstructions as to what constitutes an effective amount, from supplierssuch as Collaborative Research, Sigma Chemical Co., vascular growthfactors such as vascular endothelial growth factor (VEGF), epidermalgrowth factor (EGF), and heparin binding epidermal growth factor likegrowth factor (HB-EGF), could be incorporated into the matrix or beprovided in conjunction with the matrix. Similarly, polymers containingpeptides such as the attachment peptide RGD (Arg-Gly-Asp) can besynthesized for use in forming matrices (see e.g. U.S. Pat. Nos.4,988,621, 4,792,525, 5,965,997, 4,879,237 and 4,789,734).

In another example, the undifferentiated, partially differentiated orfully differentiated amnion-derived cells may be transplanted in a gelmatrix (such as Gelfoam from Upjohn Company) which polymerizes to form asubstrate in which the cells can grow. A variety of encapsulationtechnologies have been developed (e.g. Lacy et al., Science 254:1782-84(1991); Sullivan et al., Science 252:718-712 (1991); WO 91/10470; WO91/10425; U.S. Pat. No. 5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat.No. 4,892,538). During open surgical procedures, involving directphysical access to the damaged tissue and/or organ, all of the describedforms of undifferentiated, partially differentiated or fullydifferentiated amnion-derived cell delivery preparations are availableoptions. These cells can be repeatedly transplanted at intervals until adesired therapeutic effect is achieved.

The present invention also relates to the use of amnion-derived cells inthree dimensional cell and tissue culture systems to form structuresanalogous to tissue counterparts in vivo. The resulting tissue willsurvive for prolonged periods of time, and perform tissue-specificfunctions following transplantation into the recipient host. Methods forproducing such structures are described in U.S. Pat. Nos. 5,624,840 and6,428,802, which are incorporated herein in their entireties.

The three-dimensional matrices to be used are structural matrices thatprovide a scaffold for the cells, to guide the process of tissueformation. Scaffolds can take forms ranging from fibers, gels, fabrics,sponge-like sheets, and complex 3-D structures with pores and channelsfabricated using complex Solid Free Form Fabrication (SFFF) approaches.Cells cultured on a three-dimensional matrix will grow in multiplelayers to develop organotypic structures occurring in three dimensionssuch as ducts, plates, and spaces between plates that resemblesinusoidal areas, thereby forming new liver tissue. Thus, in preferredaspects, the present invention provides a scaffold, multi-layer cell andtissue culture system. As used herein, the term “scaffold” means athree-dimensional (3D) structure (substrate and/or matrix) that cellsgrow in or on. It may be composed of biological components, syntheticcomponents or a combination of both. Further, it may be naturallyconstructed by cells or artificially constructed. In addition, thescaffold may contain components that have biological activity underappropriate conditions. The structure of the scaffold can include amesh, a sponge or can be formed from a hydrogel.

Examples of such scaffolds include a three-dimensional stromal tissue orliving stromal matrix which has been inoculated with stromal cells thatare grown on a three dimensional support. The extracellular matrixproteins elaborated by the stromal cells are deposited onto thescaffold, thus forming a living stromal tissue. The living stromaltissue can support the growth of amnion-derived cells or differentiatedcells later inoculated to form the three-dimensional cell culture.Examples of other three dimensional scaffolds are described in U.S. Pat.No. 6,372,494.

The design and construction of the scaffolding to form athree-dimensional matrix is of primary importance. The matrix should bea pliable, non-toxic, injectable porous template for vascular ingrowth.The pores should allow vascular ingrowth. These are generallyinterconnected pores in the range of between approximately 100 and 300microns, i.e., having an interstitial spacing between 100 and 300microns, although larger openings can be used. The matrix should beshaped to maximize surface area, to allow adequate diffusion ofnutrients, gases and growth factors to the cells on the interior of thematrix and to allow the ingrowth of new blood vessels and connectivetissue. At the present time, a porous structure that is relativelyresistant to compression is preferred, although it has been demonstratedthat even if one or two of the typically six sides of the matrix arecompressed, that the matrix is still effective to yield tissue growth.

The polymeric matrix may be made flexible or rigid, depending on thedesired final form, structure and function. For repair of a defect, forexample, a flexible fibrous mat is cut to approximate the entire defectthen fitted to the surgically prepared defect as necessary duringimplantation. An advantage of using the fibrous matrices is the ease inreshaping and rearranging the structures at the time of implantation.

A sponge-like structure can also be used to create a three-dimensionalframework. The structure should be an open cell sponge, one containingvoids interconnected with the surface of the structure, to allowadequate surfaces of attachment for sufficient amnion-derived cells ordifferentiated cells to form a viable, functional implant.

The invention also provides for the delivery of amnion-derived cells,including amnion-derived cell compositions described herein, inconjunction with any of the above support matrices as well asamnion-derived membranes. Such membranes may be obtained as a by-productof the process described herein for the recovery of amnion-derivedcells, or by other methods, such as are described, for example, in U.S.Pat. No. 6,326,019 which describes a method for making, storing andusing a surgical graft from human amniotic membrane, US 2003/0235580which describes reconstituted and recombinant amniotic membranes forsustained delivery of therapeutic molecules, proteins or metabolites, toa site in a host, U.S. 2004/0181240, which describes an amnioticmembrane covering for a tissue surface which may prevent adhesions,exclude bacteria or inhibit bacterial activity, or to promote healing orgrowth of tissue, and U.S. Pat. No. 4,361,552, which pertains to thepreparation of cross-linked amnion membranes and their use in methodsfor treating burns and wounds. In accordance with the present invention,amnion-derived cells may be grown on such membranes, added to themembrane in either an undifferentiated, partially differentiated orfully differentiated form, or amnion-derived cell conditioned media orcell lysates may be added to such membranes. Alternatively, amniotictissue in which amnion-derived cells have not been stripped away may beused to deliver amnion-derived cells to a particular site. In all cases,amnion-derived cells used in conjunction with amniotic tissue or othermatrices can be used in combination with other therapeutically usefulcells and/or cells expressing biologically active therapeutics such asthose described in below.

Amnion-derived cells and cells differentiated therefrom may also be usedto humanize animal organs. Human amnion-derived cells may be similarlytransplanted into another organ such as pancreas or brain or heart. Theanimal organ may or may not be depleted of its native cells prior to thetransplant. “Humanized” organs of an animal such as a mouse, rat,monkey, pig or dog could be useful for organ transplants into humanswith specific diseases.

Humanized animal models may also be used for diagnostic or researchpurposes relating but not limited to, drug metabolism, toxicologystudies or for the production, study, or replication of viral orbacterial organisms. Mice transplanted with human hepatocytes formingchimeric human livers are currently being used for the study ofhepatitis viruses (Dandri et al. Hepatol. 33:981-988 (2001); Mercer etal. Nature Med. 7:927-933 (2001)).

Amnion-derived cells may be genetically engineered to produce aparticular therapeutic protein. Therapeutic protein includes a widerange of biologically active proteins including, but not limited to,growth factors, enzymes, hormones, cytokines, inhibitors of cytokines,blood clotting factors, peptide growth and differentiation factors.Particular differentiated cells may be engineered with a protein that isnormally expressed by the particular cell type. For example, pancreaticcells can be engineered to produce digestive enzymes. Hepatocytes can beengineered to produce the enzyme inhibitor, A1AT, or clotting factors totreat hemophilia. Furthermore, neural cells can be engineered to producechemical transmitters.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a nucleic acid encoding theprotein of interest linked to appropriate transcriptional/translationalcontrol signals. See, for example, the techniques described in Sambrooket al, 2001, “Molecular Cloning: A Laboratory Manual”; Ausubel, ed.,1994, “Current Protocols in Molecular Biology” Volumes I-III; Celis,ed., 1994.

Suitable methods for transferring vector or plasmids into amnion-derivedcells or cells differentiated therefrom include lipid/DNA complexes,such as those described in U.S. Pat. Nos. 5,578,475; 5,627,175;5,705,308; 5,744,335; 5,976,567; 6,020,202; and 6,051,429. Suitablereagents include lipofectamine, a 3:1 (w/w) liposome formulation of thepoly-cationic lipid2,3-dioleyloxy-N-[2(sperminecarbox-amido)ethyl]-N,N-d-imethyl-1-propanaminiumtrifluoroacetate (DOSPA) (Chemical Abstracts Registry name:N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxpentyl)amino)ethyl-]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanamin-trifluoroacetate),and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) inmembrane filtered water. Exemplary is the formulation Lipofectamine2000™ (available from Gibco/Life Technologies # 11668019). Otherreagents include: FuGENE™ 6 Transfection Reagent (a blend of lipids innon-liposomal form and other compounds in 80% ethanol, obtainable fromRoche Diagnostics Corp. # 1814443); and LipoTAXI™ transfection reagent(a lipid formulation from Invitrogen Corp., #204110). Transfection ofamnion-derived cells can be performed by electroporation, e.g., asdescribed in Roach and McNeish (Methods in Mol. Biol. 185:1 (2002)).Suitable viral vector systems for producing stem cells with stablegenetic alterations may be based on adenoviruses, lentiviruses,retroviruses and other viruses, and may be prepared using commerciallyavailable virus components.

Amnion-derived cells that are undifferentiated, partiallydifferentiated, or fully differentiated may be administered ortransplanted to a subject to provide various cellular or tissuefunctions specific to the differentiated cell type. For example,amnion-derived cells differentiated into hepatocytes may be transplantedinto a patient suffering from liver disease. The progress of therecipient receiving such cells or transplants can be determined usingassays that include blood tests known as liver function tests. Efficacyof treatment can be determined by immunocytochemical staining for livercell markers, microscopic determination of whether canalicularstructures form in growing tissue, and the ability of the treatment torestore synthesis of liver-specific proteins. Amnion-derivedcell-derived hepatocytes of the invention can be assessed in animalmodels for their ability to repair liver damage. Hepatocytes derivedfrom amnion-derived cells may be used in assays to detect the activityof specific metabolic pathways. For detailed examples of the above, seeUS2003/0235563 and US2004/0161419, which are incorporated herein byreference.

Pancreatic cells derived from amnion-derived cells can be usedtherapeutically for treatment of various diseases associated withinsufficient functioning of the pancreas. Pancreatic diseases andtreatment thereof using the pancreatic cells derived from amnion-derivedcells of the subject invention are described in more detail below.

The present invention also provides for administration of neural cellsderived from amnion-derived cells for treatment of neurological disease.Neurological disease refers to a disease or condition associated withany defects in the entire integrated system of nervous tissue in thebody: the cerebral cortex, cerebellum, thalamus, hypothalamus, midbrain,pons, medulla, brainstem, spinal cord, basal ganglia and peripheralnervous system. Examples include but are not limited to: Parkinson'sdisease, Huntington's disease, Multiple Sclerosis, Alzheimer's disease,amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), Musculardystrophy, choreic syndrome, dystonic syndrome, stroke, and paralysis.

The amnion-derived cells may be used in in vitro priming procedures thatresult in neural stem cells becoming neurons when grafted intonon-neurogenic or neurogenic areas of the CNS. For details and examples,see US2003/0235563 and US2004/0161419, both which are incorporatedherein by reference.

The amnion-derived cells can be used to produce vascular endothelialcells that may be used in methods for remodeling tissue or replacing ascar tissue in a subject. Vascular endothelial cells may also be used torepair vascular damage.

In an exemplary embodiment, a pharmaceutical composition comprising aneffective amount of the vascular endothelial cells may be used to treata subject with a vascular disease. Vascular disease refers to a diseaseof the human vascular system. Examples include peripheral arterialdisease, abdominal aortic aneurysm, carotid disease, and venous disease.

The present invention also provides for cardiomyocytes derived fromamnion-derived cells, which may be used therapeutically for treatment ofvarious diseases associated with cardiac dysfunction. Cardiac disease orcardiac dysfunction as used herein refers to diseases that result fromany impairment in the heart's pumping function. This includes, forexample, impairments in contractility, impairments in ability to relax(sometimes referred to as diastolic dysfunction), abnormal or improperfunctioning of the heart's valves, diseases of the heart muscle(sometimes referred to as cardiomyopathy), diseases such as angina andmyocardial ischemia and infarction characterized by inadequate bloodsupply to the heart muscle, infiltrative diseases such as amyloidosisand hemochromatosis, global or regional hypertrophy (such as may occurin some kinds of cardiomyopathy or systemic hypertension), and abnormalcommunications between chambers of the heart (for example, atrial septaldefect). For further discussion, see Braunwald, Heart Disease: aTextbook of Cardiovascular Medicine, 5th edition, W B Saunders Company,Philadelphia Pa. (1997) (hereinafter Braunwald). Cardiomyopathy refersto any disease or dysfunction of the myocardium (heart muscle) in whichthe heart is abnormally enlarged, thickened and/or stiffened. As aresult, the heart muscle's ability to pump blood is usually weakened.The disease or disorder can be, for example, inflammatory, metabolic,toxic, infiltrative, fibroblastic, hematological, genetic, or unknown inorigin. There are two general types of cardiomyopathies: ischemic(resulting from a lack of oxygen) and nonischemic. Other diseasesinclude congenital heart disease which is a heart-related problem thatis present since birth and often as the heart is forming even beforebirth or diseases that result from myocardial injury which involvesdamage to the muscle or the myocardium in the wall of the heart as aresult of disease or trauma. Myocardial injury can be attributed to manythings such as, but not limited to, cardiomyopathy, myocardialinfarction, or congenital heart disease.

The amnion-derived cells and/or differentiated cardiomyocytes may beadministered and/or transplanted to a subject suffering from a cardiacdisease in any fashion as previously discussed.

Methods are also provided for screening agents that affect cardiomyocytedifferentiation or function. For details and examples see US2003/0235563and US2004/0161419, which are incorporated herein by reference.

In another embodiment, amnion-derived cells, and their derivatives, canbe used to screen various compounds to determine the effect of thecompound on cellular growth, proliferation or differentiation of thecells. Methods of measuring cell proliferation are well known in the artand most commonly include determining DNA synthesis characteristic ofcell replication. There are numerous methods in the art for measuringDNA synthesis, any of which may be used according to the invention. Forexample, DNA synthesis may be determined using a radioactive label(3H-thymidine) or labeled nucleotide analogues (BrdU) for detection byimmunofluorescence. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the compound. A control assay can also be performed toprovide a baseline for comparison. Identification of the amnion-derivedcell population(s) amplified in response to a given test agent can becarried out according to such phenotyping as described above.

In order to assess the effect of a test agent on amnion-derived celldifferentiation or function, the agent may be contacted with theamnion-derived cells and differentiation assessed using any means knownto one of skill in the art. For examples and details, see US2003/0235563and US2004/0161419, which are incorporated herein by reference.

In another embodiment, amnion-derived cell compositions prepared asdescribe herein are used as feeder layers for the growth of embryonicstem cells. Such amnion-derived cell compositions are preferablyanimal-free. Examples of use of such cells as feeders layers can befound in Miyamoto, K., et al. Stem Cells 2004:22:433-440 which isincorporated by reference in its entirely herein.

Wound healing—The compositions and methods of the present invention areeffective in accelerating wound healing of wounds caused by a number ofsources, including but not limited to incisional, compression, thermal,acute, chronic, infected, and sterile injuries. The instant invention isbased upon the discovery that undifferentiated, partially differentiatedor fully differentiated amnion-derived cells, conditioned mediumtherefrom, cell lysates therefrom, extracellular matrices therefrom,alone or in combination, as well as composition of placental-derivedcells as defined herein can accelerate the wound healing process for allwound types, particularly when administered topically, i.e. to thesurface of the wound site. Using amnion-derived cells and/or conditionedmedium from such amnion-derived cells, all wound types, mechanical orthermal, acute or chronic, infected or sterile, undergo healing morerapidly than similar wounds left to heal naturally or which are treatedwith currently available methods. A “therapeutically effective amount”of a therapeutic agent within the meaning of the present invention willbe determined by a patient's attending physician or veterinarian. Suchamounts are readily ascertained by one of ordinary skill in the art andwill enable accelerated wound healing when administered in accordancewith the present invention. Factors which influence what atherapeutically effective amount will be include, the specific activityof the therapeutic agent being used, the wound type (mechanical orthermal, full or partial thickness, etc.), the size of the wound, thewound's depth (if full thickness), the absence or presence of infection,time elapsed since the injury's infliction, and the age, physicalcondition, existence of other disease states, and nutritional status ofthe patient. Additionally, other medication the patient may be receivingwill effect the determination of the therapeutically effective amount ofthe therapeutic agent to administer.

In addition, compositions of the invention may play a role in moresubstantial wound healing, such as in the regeneration of limbs.US2003/0212024, which is incorporated by reference herein, sets forthmethods of testing for such ability by measuring regeneration in thezebrafish, which is capable of complete regeneration followingamputation of the distal fin. Following amputation, completeregeneration occurs in several steps, including formation of a woundepidermis, migration of fibroblasts and scleroblasts (or osteoblasts)toward the wound epidermis, formation of a blastema, and outgrowth ofthe blastema via cell division and differentiation of the proximalportion of the fin to form specific structures of the regenerated fin.

In a preferred embodiment of the present invention, amnion-derived cellsand/or conditioned medium therefrom, and/or cell lysates thereof shouldbe topically administered to the wound site to promote accelerated woundhealing in the patient. This topical administration can be as a singledose or as repeated doses given at multiple designated intervals. Itwill readily be appreciated by those skilled in the art that thepreferred dosage regimen will vary with the type and severity of theinjury being treated.

Formulations suitable for topical administration in accordance with thepresent invention comprise therapeutically effective amounts of thetherapeutic agent with one or more pharmaceutically acceptable carriersand/or adjuvants. Amnion-derived cells, conditioned media therefrom andcell lysates thereof may be used in conjunction with a variety ofmaterials routinely used in the treatment of wounds, such as collagenbased creams, films, microcapsules, or powders; hyaluronic acid or otherglycosaminoglycan-derived preparations; creams, foams, suture material;and wound dressings. Alternatively, the amnion-derived cell compositionscan be incorporated into a pharmaceutically acceptable solution designedfor topical administration.

Cosmetic Applications—The same properties that make undifferentiated,partially differentiated or fully differentiated amnion-derived cells,conditioned medium therefrom, cell lysates therefrom, extracellularmatrices therefrom, alone or in combination, as well as composition ofplacental-derived cells as defined herein useful for wound healing makethem similarly well-suited for the treatment of cosmetic and/ordermatological conditions, including aging skin. The dermal layer ofskin, important in maintaining the elasticity and appearance of theskin, thins with age, leading to sagging and wrinkles.

As described above, fetal skin has much more effective repairmechanisms, and, once wounded, it is able to heal without the formationof scars. This capability does appear to require the fetal immunesystem, fetal serum, or amniotic fluid (Bleacher J C, et al., J PediatrSurg 28: 1312-4, 1993); Ihara S, Motobayashi Y., Development 114:573-82. 1992). Such abilities of fetal tissue have led to the suggesteduse of compounds produced by fetal tissue for regenerating and/orimproving the appearance of skin (see, for example, US 2004/0170615,which is incorporated by reference in its entirety herein).

The present invention contemplates the use of the amnion-derived cellcompositions described herein, as well as conditioned medium therefrom,and cell lysates thereof, in the use of novel cosmetic skin carecompositions. Such compounds may be delivered to skin by way of, but notlimited to, a solution, a lotion, an ointment, a cream, a gel, or a skinpeelable strip.

The methods generally include the step of topically applying a safe andeffective amount of the composition to the skin of a mammal in needthereof. Additional skin care components, as well as cosmeticallyacceptable, dermatologically acceptable or pharmaceutically acceptablecarriers may be included in such compositions.

Cosmetic compositions usually comprise an aqueous phase that is gelled,i.e. thickened, using one or more thickener(s) or gelling agent(s).These may be, for example, lotions which are aqueous solutions notcontaining an oily phase, or emulsions which may be direct oil-in-wateremulsions including a fatty phase or oily phase dispersed in an aqueouscontinuous phase, or water-in-oil reverse emulsions including an aqueousphase dispersed in an oily continuous phase. The term “emulsions” meansherein both the dispersions obtained in the absence of emulsifyingsurfactants and the emulsions obtained in the presence of emulsifyingsurfactants.

Oil-in-water emulsions are the emulsions most frequently sought incosmetics due to the fact that, when applied to the skin, they give asofter, less greasy, fresher and lighter feel than water-in-oil emulsionsystems, by virtue of the presence of water in the continuous outerphase.

The nature of the compounds used for gelling the aqueous phase and theircontent in the composition are chosen as a function of the desired typeof texture, which may range from fluid lotions to more or less thickemulsions that may constitute milks or creams. The main thickeners orgelling agents used in cosmetics are chosen from the following compoundsnatural polymers such as xanthan gum and guar gum or cellulosederivatives, starches and alginates and crosslinked polymeric gellingagents such as the Carbopols or crosslinked and at least partiallyneutralized 2-acrylamido-2-methylpropanesulfonic acid polymers.

Hearing Loss—Undifferentiated, partially differentiated or fullydifferentiated amnion-derived cells, conditioned medium therefrom, celllysates therefrom, extracellular matrices therefrom, alone or incombination, as well as composition of placental-derived cells asdefined herein may also be used to treat hearing loss. Heretofore,hearing loss has been considered incurable because hair cells, which arethe sensory cells of the cochlea, do not regenerate. Recently, however,both embryonic and adult stem cells have been shown to be capable ofdifferentiating into mechanosensory hair cells (Li, H. et al. NatureMed. 9:1293-1299 (2003); Li, H. et al. Proc. Natl. Acad. Sci. USA100:13495-13500). In addition, Atoh1 gene therapy has been used topromote phenotypic transgeneration, thus promoting the generation ofhair cells from nonsensory cells in deaf mammals (Izumikawa, M. et al.2005 Nature Medicine 11 (3):271-276). Further, human amniotic epithelialcells transplanted into the inner ear of guinea pigs have been shown tosurvive for up to three weeks and express crucial proteins which maymaintain homeostasis (Yuge, I. et al. (2004):77(9)1452-1471).

Methods of Differentiating Amnion-Derived Cells and Differentiated CellTypes

The amnion-derived cells may be contacted with various growth factors(termed differentiation factors) that influence differentiation of suchstem cells into particular cell types such as hepatocytes, pancreaticcells, vascular endothelial cells, muscle cells, cardiomyocytes andneural cells. For examples, see US2003/0235563 and US2004/0161419, thecontents of which are incorporated herein by reference).

The literature is replete with additional differentiation protocols forembryonic as well as non-embryonic stem or other multipotent cells,including stem cells. For example, U.S. Pat. Nos. 6,607,720 and6,534,052 described methods of improving cardiac function usingembryonic stem cells and genetically altered embryonic stem cells inwhich differentiation has been initiated, for improving cardiac functionand repairing heart tissue. U.S. Pat. No. 6,387,369 provides methods ofcardiac tissue and muscle regeneration using mesenchymal stem cells.Shin, S. et al. have recently reported the differentiation of embryonicstem cells into motor neurons using a combination of basic fibroblastgrowth factor, sonic hedgehog protein, and retinoic acid. (Human motorneuron differentiation from human embryonic stem cells. Stem Cells Dev.2005 Jun.; 14(3):266-9). All of these references are incorporated hereinin their entirety. One skilled in the art will recognize that any ofthese protocols can be applied to the amnion-derived cell compositionsdescribed herein to produce partially or fully differentiated cells forsuch uses. Other exemplary protocols are set forth below:

Endoderm (pancreatic differentiation). Amnion-derived cell are exposedto conditions for differentiation of an islet progenitor cell populationexpressing PDX 1. Briefly, cells are initially exposed to antagonists ofthe Sonic Hedgehog (SHh) signaling pathway to promote endodermdifferentiation. Subsequent differentiation to early islet progenitorcells is accomplished using a combination of factors and conditions thatpromote cessation of cell growth, aggregation of differentiating cells,and expression of early pancreatic determination genes. Cells areharvested for RNA and analyzed by reverse transcriptase-PCR(RT-PCR) forSox-17 and PDX 1.

Mesoderm (cardiac differentiation). Clusters of cells taken from thesuspension cultures are transferred to gelatin- or poly-L-lysine coatedplates for 8 days in culture medium with serum (80% KO-DMEM, 1 mMglutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, and20% FBS). For Days 2-4 in this culture 1 or 10 μM 5-aza-2′-deoxycytidineare added to the medium (Xu, C. et al. (2002) Circ. Res. 91:501-508).Analysis is performed on Day 8. Cells are harvested for RNA and analyzedby reverse transcriptase-PCR(RT-PCR) for GATA-4, Nkx2.5 and MEF-2. Thesetranscription factors are expressed in precardiac mesoderm and persistin cardiac development.

Ectoderm (neural differentiation). Clusters are removed from thelarge-scale apparatus and transferred to ultra-low adherence 6-wellplates. The differentiation protocol described by Carpenter, M. K. etal. (2001) Exp Neurol 172:383-397 for human embryonic stem cells isfollowed for differentiation as follows. 10 mM all-trans retinoic acid(RA) will added to the culture medium (80% KO-DMEM, 1 mM glutamine, 0.1mM β-mercaptoethanol, 1% non-essential amino acids, and 20% FBS)containing these clusters in suspension. After 4 days in suspension,clusters are plated onto poly-L-lysine/fibronectin-coated plates indifferentiation medium (DMEM/F-12 with B27 (Gibco), 10 ng/ml humanepidermal growth factor (hEGF), 10 ng/ml human basic fibroblast growthfactor (hbFGF) (Gibco), 1 ng/ml human platelet-derived growth factor-AA(hPDGF-AA) (R & D Systems), and 1 ng/ml human insulin-like growthfactor-1 (hIGF-1) (R & D Systems) for 3 days. After 3 days under theseconditions, the cells are harvested for RNA or fixed. Fixed cells areimmunostained for nestin, polysialylated neural cell adhesion molecule(PS-NCAM), and A2B5. RNA is analyzed by reversetranscriptase-PCR(RT-PCR) for nestin, GFAP and MAP-2.

Differentiated cells derived from amnion-derived cells may be detectedand/or enriched by the detection of tissue-specific markers byimmunological techniques, such as flow immunocytochemistry forcell-surface markers, immunohistochemistry (for example, of fixed cellsor tissue sections) for intracellular or cell-surface markers, Westernblot analysis of cellular extracts, and enzyme-linked immunoassay, forcellular extracts or products secreted into the medium. The expressionof tissue-specific gene products can also be detected at the mRNA levelby Northern blot analysis, dot-blot hybridization analysis, or byreverse transcriptase initiated polymerase chain reaction (RT-PCR) usingsequence-specific primers in standard amplification methods.

Alternatively, differentiated cells may be detected using selectionmarkers. For example, amnion-derived cells can be stably transfectedwith a marker that is under the control of a tissue-specific regulatoryregion as an example, such that during differentiation, the marker isselectively expressed in the specific cells, thereby allowing selectionof the specific cells relative to the cells that do not express themarker. The marker can be, e.g., a cell surface protein or otherdetectable marker, or a marker that can make cells resistant toconditions in which they die in the absence of the marker, such as anantibiotic resistance gene (see e.g., in U.S. Pat. No. 6,015,671).

Pancreatic Progenitor Cells

In another embodiment of the invention, cells are treated such that theydifferentiate into pancreatic progenitor cells, In this embodiment,amnion-derived cells, non-insulin producing embryonic, neonatal or fetalcells are cultured in serum-free culture medium comprising a SHhantagonist such as cyclopamine or jervine to obtain pancreatic cellshaving the identifying characteristics of endoderm, which include butare not limited to protein expression of HNF1α, HNF1β, HNF4α, HNF6,Fox2a and PDX1. The cells may be cultured in such medium after culturingin basal medium.

Pancreatic progenitor cells of the present invention expressing PDX1protein in the nucleus may also be obtained by culturing cells in mediumcomprising an SHh antagonist. The cells may subsequently be cultured inmedium comprising TAT-PDX1 fusion protein. Procedures for obtainingTAT-PDX1 are described infra. In a preferred embodiment, at least 20% ofthe cells in the composition, culture or population of the presentinvention express PDX1 protein in the nucleus. In other embodiments, atleast 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in thecomposition, culture or population of the present invention express PDX1protein in the nucleus. In a specific, preferred embodiment, 100% of thecells express PDX1 protein in the nucleus.

The composition, culture or population of cells of the present inventionmay be cultured in suspension or on a solid support, such as an adherentmatrix or substrate. Details of such solid supports are described above.In one embodiment, the composition of cells is cultured on a Matrigellayer. Matrigel (Collaborative Research, Inc., Bedford, Mass.) is acomplex mixture of matrix and associated materials derived as an extractof murine basement membrane proteins, consisting predominantly oflaminin, collagen IV, heparin sulfate proteoglycan, and nidogen andentactin, and was prepared from the EHS tumor (Kleinman et al, (1986)Biochemistry 25: 312-318). Other such matrices can be provided, such asHumatrix. Likewise, natural and recombinantly engineered cells can beprovided as feeder layers to the instant cultures. In anotherembodiment, the culture vessels are coated with one or moreextra-cellular matrix proteins including, but not limited to,fibronectin, superfibronectin, laminin, collagen, and heparin sulfateproteoglycan.

In another embodiment, the progenitor cells of the present invention arecultured in the presence of three dimensional matrices. Examples of suchthree dimensional matrices are described in detail above.

Pancreatic Progenitor Cell Nuclei—The invention is further directed tothe nuclei of the pancreatic progenitor cells of the present invention.The nuclei of these cells may be obtained using methods known in theart. These include removing the membranes from cells by eithermechanical disruption or chemical means such as treatment withhyaluronidase or performed by mechanically extracting the nucleus with apipet and inserting it into a different or similar cell that has had itsnucleus removed.

The nuclei may then subsequently be transferred into somatic or germcells by, for example, intracytoplasmic injection, chemical fusion orelectrofusion using methods known in the art as described in, forexample, US 20030234430 or US 20040268422.

In a particular embodiment, therapeutic cloning may be undertaken usingthese nuclei to obtain cells that can be used for endoderm or other celldifferentiation. In a more particular embodiment, the nuclei may be usedto obtain embryonic stem-like cells. Details concerning therapeuticcloning are described above.

In addition to germ cells, the recipient cell may be any mammalian cell.In one embodiment, the mammalian cell is enucleated prior to receivingthe donor nucleus. In another embodiment, the mammalian cell in notenucleated prior to receiving the donor nucleus. In this embodiment,both the recipient and donor nuclei are present in the recipient cell.The nuclei may fuse or they may stay separate. Instances in which it isdesirable for both recipient and donor nuclei to be present are ones inwhich the object is to confer the tissue-specific functionality of thedonor cell onto the recipient cell while still maintaining thetissue-specific functionality of the recipient cell. One of skill in theart will recognize that other combinations are within the scope of theinvention.

Detection of Pancreatic Cells—The pancreatic cells of the presentinvention described hereinabove can be detected in the composition,culture or population of cells of the present invention by detecting thepresence or absence of various markers, such as HNF1α, HNF1β, HNF4α,HNF6, Foxa2, PDX1, Nkx2.2, Nkx6.1, Sox17, Cerberus, Hesx1, LeftyA, Otx1and/or Otx2, insulin, human C-peptide, somatostatin and islet-1. In oneembodiment, fragments of HNF1α, HNF1β, HNF4α, HNF6, Foxa2, PDX1, Nkx2.2,Nkx6.1, Sox17, Cerberus, Hesx1, LeftyA, Otx1 and/or Otx2, insulin, humanC-peptide, somatostatin or islet-1 may be used as probes or primers fordetecting RNA transcription of the markers. The markers may be detectedby Northern blot analysis, for example, by hybridizing either total orpoly A RNA isolated from the cells with probes and primers between10-500 nucleotides in length, preferably between 20-200 nucleotides inlength, more preferably between 20-100 nucleotides in length and mostpreferably between 20-50 nucleotides in length and subjecting to agarosegel electrophoresis. Alternatively, these fragments may be used asRT-PCR primers between about 10-100 nucleotides in length to amplify theRNA isolated from the cells. Cells suitable for such an analysis includecells isolated from human tissue. Methods for performing primer-directedamplification (routine or long range PCR) are well known in the art(see, for example, PCR Basics: From Background to Bench, Springer Verlag(2000); Gelfand et al., (eds.), PCR Strategies, Academic Press (1998)).Such probes may be between 20-5000 nucleotides in length and maypreferably be between 20-50 nucleotides in length.

Alternatively, the above-mentioned markers may be detected usingantibodies to the markers in RIA, ELISA, Western Blot orimmunocytochemical techniques. The invention is thus directed to kitscomprising antibodies binding to two or more of the above-mentionedmarkers.

TAT-PDX1 Fusion Protein—The invention also relates to TAT-PDX1 fusionprotein used in the culture medium for the purpose of obtainingpancreatic progenitor cells comprising PDX1 protein in the nucleus ofthe progenitor cells. TAT-PDX1 has the formula:

Wherein TAT is a TAT peptide having a self cell penetration property.The TAT peptide is derived from human immunodeficiency virus type-1 andis capable of passing through a cell membrane to easily penetrate thecell. This property is thought to be due to the protein transductiondomain in the middle region of the TAT peptide sequence. R1 may be sidechains of glutamine, lysine, arginine and/or glycine and n is an integerof 4 to 12.

In a specific embodiment, the TAT peptide may beArg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg;Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys; orArg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg.

PDX1 is a homeodomain-containing protein and is thought to be a keyregulator of islet development and insulin gene transcription in betacells (Inoue et al., 1996, Diabetes 6:789-794). It has the followingamino acid sequence:

     MNGEEQYYAATQLYKDPCAFQRGPAPEFSASPPACLYMGRQPPPPPPHPFPGALGAEQGSPPDISPYEVPPLADDPAVAHLHHHLPAQLALPHPPAGPFPEGAEPGVLEENRVQLPFPWMKSTKAHAWKGQWAGGAYAAEPEENKRTRTAYTRAQLLELEKEFLFNKYISRPRRVELAVMLNLTERHIKIWFQNRRMKWKKEEDKKRGGGTAVGGGGVAEPEQDCAVTSGEELLALPPPPPPGGAVPPAAPVAAREGRLPPGLSASPQPSSVAPRRPQEPR

and is encoded by the following nucleotide sequence

           gccctgtgtc gcccgcaggc ggcgcctacg ctgcggagcc ggaggagaacaagcggacgc gcacggccta cacgcgcgca cagctgctag agctggagaa ggagttcctattcaacaagt acatctcacg gccgcgccgg gtggagctgg ctgtcatgtt gaacttgaccgagagacaca tcaagatctg gttccaaaac cgccgcatga agtggaaaaa ggaggaggacaagaagcgcg gcggcgggac agctgtcggg ggtggcgggg tcgcggagcc tgagcaggactgcgccgtga cctccggcga ggagcttctg gcgctgccgc cgccgccgcc ccccggaggtgctgtgccgc ccgctgcccc cgttgccgcc cgagagggcc gcctgccgcc tggccttagcgcgtcgccac agccctccag cgtcgcgcct cggcggccgc aggaaccacg atgagaggcaggagctgctc ctggctgagg ggcttcaacc actcgccgag gaggagcaga gggcctaggaggaccccggg cgtggaccac ccgccctggc agttgaatgg ggcggcaatt gcggggcccaccttagaccg aaggggaaaa ccc

The entire PDX 1 sequence may be used in the fusion protein of thepresent invention. Alternatively, a fragment of the PDX1 having PDX1activity (e.g., regulation of insulin transcription, regulation of PDX1transcription, regulation of Nkx2.2 transcription) may be used. Anon-limiting example of such a fragment would be a peptide sequenceencompassing the homeobox domain and comprising the sequence:

     NKRTRTAYTRAQLLELEKEFLFNKYISRPRRVELAVMLNLTERHIKIWFQNRRMKW KKEE

The PDX1 peptide may contain conservative amino acid substitutions thatdo not significantly affect the folding and/or activity of the protein;small deletions, typically of one to about 30 amino acids; small amino-or carboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain. Examples of conservative substitutions are withinthe group of basic amino acids (arginine, lysine and histidine), acidicamino acids (glutamic acid and aspartic acid), polar amino acids(glutamine and asparagine), hydrophobic amino acids (leucine, isoleucineand valine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine, threonineand methionine). Amino acid substitutions which do not generally alterthe specific activity are known in the art and are described, forexample, by H. Neurath and R. L. Hill, 1979, In, The Proteins, AcademicPress, New York. The most commonly occurring exchanges are Ala/Ser,Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly,Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, as well as thesein reverse. Alternatively, the nucleotide sequence encoding PDX1 maycontain alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred.

The fusion protein or peptide may be obtained using recombinant DNAmethods. For example, a nucleic acid sequence encoding PDX1 or PDX1peptide may be inserted into a vector containing nucleic acid sequencesencoding the TAT peptide. In a particular embodiment, the TAT sequenceis obtained by PCR and inserted, for example, into a pET vector, orother protein expression vector capable of expressing a fusion protein.The fusion protein is expressed, isolated and purified using proceduresknown in the art, such as HPLC and column chromatography.

Alternatively, the fusion protein may be produced by solid phasesynthesis using an organosynthesizer for peptide synthesis. The methodis Merrifield solid-phase peptide synthesis (J. Am. Chem. Soc. 85,2149-2154 (1963)). The peptide is synthesized by sequentially couplingan alpha-amino protected amino acid to an amino terminal of a peptidechain attached to a solid support resin after activation. Aftersynthesis, the peptide is cut from the resin, and the protecting groupis removed with a reagent such as trifluoroacetic acid (TPA). Thepeptide is separated from the TFA solution by filtration,centrifugation, or extraction with diethylether, and it can be purifiedby high performance liquid chromatography (HPLC) or other methods.

In addition, other TAT fusion proteins may be made. For example,TAT-PDX1, TAT-Hblx9, TAT-Ngn3, TAT-p48, or TAT-Foxa2 may be used inpracticing the methods of the invention. Such fusion proteins made bemade using the methods described above.

Uses of Pancreatic Progenitor Cells—The pancreatic progenitor cells ofthe present invention and compositions thereof can be usedtherapeutically for treatment of various diseases associated withinsufficient functioning of the pancreas. As used herein, the term“pancreatic disease” may include but is not limited to pancreaticcancer, insulin-deficiency disorder such as Insulin-dependent (Type 1)diabetes mellitus (IDDM) and Non-insulin-dependent (Type 2) diabetesmellitus (NIDDM), hepatitis C infection, exocrine and endocrinepancreatic diseases.

The progenitor cells of the present invention can be used to producepopulations of differentiated pancreatic cells for repair subsequent topartial pancreatectomy, e.g., excision of a portion of the pancreas.Likewise, such cell populations can be used to regenerate or replacepancreatic tissue loss due to, pancreatolysis, e.g., destruction ofpancreatic tissue, such as pancreatitis, e.g., a condition due toautolysis of pancreatic tissue caused by escape of enzymes into thetissue. Pancreatic cells may be transplanted into the pancreas or toectopic sites, such as, but not limited to the liver, portal vein,spleen, mammary gland, kidney or at or near the intestines. In oneembodiment the cells of the invention may be administeredsubcutaneously.

Methods of administration include encapsulating differentiated betaislet cells producing insulin in implantable hollow fibers. Such fiberscan be pre-spun and subsequently loaded with the differentiated betaislet cells of the invention (see U.S. Pat. No. 4,892,538; U.S. Pat. No.5,106,627; Hoffman et al. Expt. Neurobiol. 110:39-44 (1990); Jaeger etal. Prog. Brain Res. 82:41-46 (1990); and Aebischer et al. J. Biomech.Eng. 113:178-183 (1991)), or can be co-extruded with a polymer whichacts to form a polymeric coat about the beta islet cells (U.S. Pat. No.4,391,909; U.S. Pat. No. 4,353,888; Sugamori et al. Trans. Am. Artif.Intern. Organs 35:791-799 (1989); Sefton et al. Biotechnol. Bioeng.29:1135-1143 (1987); and Aebischer et al. Biomaterials 12:50-55 (1991)).

The cells of the present invention may be genetically engineered toproduce a particular therapeutic protein. As used herein the term“therapeutic protein” includes a wide range of biologically activeproteins including, but not limited to, growth factors, enzymes,hormones, cytokines, inhibitors of cytokines, blood clotting factors,peptide growth and differentiation factors. Particular differentiatedcells may be engineered with a protein that is normally expressed by theparticular cell type. In a particular embodiment, pancreatic cells canbe engineered to produce digestive enzymes.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a nucleic acid encoding theprotein of interest linked to appropriate transcriptional/translationalcontrol signals and are described in more detail above.

The pancreatic progenitor cells and compositions, populations andcultures of the present invention may be used to determine if a testagent is toxic to a pancreatic cell by contacting the cells of thepresent invention with an appropriate amount of the test agent for atime sufficient for a toxic effect on the pancreatic cell to be detectedand determining whether the test agent has a toxic effect on thepancreatic cell.

The pancreatic progenitor cells differentiated therefrom may also beused to humanize animal organs. Human amnion-derived cells may besimilarly transplanted into another organ such as pancreas or brain orheart. The animal organ may or may not be depleted of its native cellsprior to the transplant.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Preparation of Amnion-Derived Cell Compositions

Recovery of amnion-derived cells—Amnion-derived cells were dissociatedfrom starting amniotic membrane using the dissociation agents PXXIII,and trypsin. The average weight range of an amnion was 18-27 g. Thenumber of cells recovered per g of amnion was about 10−15×10⁶ fordissociation with PXXIII and 5−8×10⁶ for dissociation with trypsin.

Culture conditions—The primary amnion-derived cells were cultured for 5passages in the following media: Stemline II+10% FBS, Stemline II+10%plasbumin (pb), Ultraculture+10% plasbumin (pb), and DMEM+10% FBS. Eachculture condition was tested using 15 million cells/g amnion, 10 millioncells/g amnion, and 5 million cells/g amnion, depending on the enzymeused for recovery of the primary cells. For instance, using PXXIII, 15million cells/g amnion were obtained, while using trypsin, 10 millioncells/g amnion were obtained, while other enzymes resulted in evenlesser recovery (5 million cells/g amnion).

Passaging—Cells were passaged 5 times as follows: The cells were grownattached to a culture flask (on tissue culture treated plastic). Thecells were left to divide and grow. The cells were removed from theplastic using “tryple” (Gibco), a trypsin-like product that isanimal-free GMP grade. Once unattached, the cells were centrifuged, andthe cell pellet removed and resuspended in the culture medium withprotein and additives (10 ng/ml EGF) and replated back onto freshflasks. Cells were grown in a humidified atmosphere at 37° C. and 5%CO₂.

Results. The results are show in Table 1 below. The data are reported asamnion-derived cells×10⁶/gram of amnion.

TABLE 1 Starting Isolation Efficiency 5 mill/g 10 mill/g 15 mill/gStemline + 10% pb 1363 2726 4089 Stemline + 10% FBS 1024 2048 3072Ultraculture + 10% pb 575 1151 1726 DMEM + 10% FBS 128 256 384 DMEM +10% pb 391 783 1174

The results indicate that the use of either Stemline or Ultraculturewith added plasbumin (pb) or albumin, the primary cultures are expandedto a level that is at least 4 fold and as much as 10 fold higher than isobtained using previous methodology (DMEM with fetal bovine serum). Eventhe use of plasbumin (pb) in the basal media DMEM resulted in anexpanded amnion-derived cell composition, having a 3-fold increase inmultipotent cells as compared to the previous method of using DMEM withfetal bovine serum.

Another significant result observed was that cells grown in mediumcontaining plasbumin displayed a spheroidal phenotype after passaging.When the amnion-derived cells were removed from the tissue culturesurface with the digestive enzyme and replated, amnion-derived cellsformed small clusters of cells that were not firmly adhered to theculture surface. Some of the clusters of cells were completely insuspension. These amnion-derived cell clusters proliferated until up to200 cells were present in the clusters. After a period of 1-5 days, theclusters of cells reattached and flattened out to form an adherentmonolayer. This clustering phenotype was observed at each passage.Further studies indicated that such clustering occurs in the followingmedia containing either recombinant human albumin, plasbumin, orplasmanate: OptiPRO SFM, VP-SFM, Iscove's MDM, HPGM, UltraMDCK, StemlineII and Stemline I, DMEM, and DMEM:F12, but not in Advanced DMEM,Knockout DMEM, 293 SFM II, Pro 293S-CDM, Pro 293A-CDM orUltracultureVP-SFM.

Example 2 Scale-Up of Amnion-Derived Cells on Microcarrier Beads inSpinner Flasks

Methods—One of the most common and oldest techniques for maintainingcells in suspension culture is by the use of spinner flasks. The cellscan be either attached to microcarrier beads (adherent) or growingcompletely without any surface attachment (non-adherent). In eithercase, these flasks consist of a sterile vessel that contains a magneticstirring mechanism that permits continuous stirring of the medium andcells under sterile conditions. This continuous stirring facilitates thediffusion of nutrients, promotes oxygenation of the medium, andeliminates concentration gradients. The vessels are stirred in atemperature-controlled, CO₂ incubator.

Amnion-derived cells are an epithelial cell type that areanchorage-dependent which may interfere with or prevent their adaptationto a pure suspension system. Although amnion-derived cells may survivein suspension culture, the proliferation of these cells may not beoptimal without any substrate for attachment and/or many of the cells insuspension may undergo preliminary differentiation. One method ofaddressing this while maintaining the ability to grow the cells in a3-dimensional system is to grow the cells on microcarrier beads.Microcarriers are typically small (30-1000 μm diameter) glass,polystyrene or dextran beads with a surface treatment to enhanceattachment. The microcarriers provide the advantage of a very largesurface area to which the cells attach allowing for culture at very highdensities in a minimal volume of medium. For example, 1 gram dry weightof typical microcarriers is equal to 2000 cm² of surface area. A smallnumber of microcarriers in cell culture medium can support the growth ofsignificant numbers of anchorage-dependent cells.

Adherent culture: Amnion-derived cells isolated from 3 differentplacentas were placed into either spinner flasks with microcarrier beads(adherent cells) or normal T-flasks (control static adherent cells) andincubated at 37° C. at 5% CO₂ in air. The cells and beads were seededinto the spinner flasks at a ratio of 1 g of beads per 66×10̂6 cells.10×10̂6 cells were plated into each T-flask. Periodically, cells werecounted, and viability assessed, using a Guava PCA-96 Personal CellAnalyzer (ViaCount package). As little as 20 μl of sample was used forthis analysis. A graph of total and viable cell counts per ml wasblotted with time to ensure that the cells were able to divide andremain viable over time in culture.

Results—In all three experiments, amnion-derived cells were shown to becapable of proliferating to at least 1.5 times seeding density, thusdemonstrating that microcarrier bead spinner flask culture methods are afeasible alternative to static culture.

Example 3 Scale-Up of Amnion-Derived Cell Compositions in Suspension

Amnion-derived cells were cultured in ultra-low adherence tissue culture6-well plates (Corning) in various mammalian cell culture media. Theseculture media were selected on the basis of their ability to promoteproliferation of other mammalian cell types in suspension culture (i.e.293S, Ultraculture, Opti-MEM). Additives to the culture medium in theseexperiments include a proprietary source of protein, and EGF (10-20ng/ml) which preliminary experiments show is required for proliferationof amnion-derived cells. Amnion-derived cells were plated at a densityof 1.3×10⁶ cells/well, and the cultures were maintained at 37° C., in 5%CO₂ in air. Culture medium was replaced every two days and cell numberwas assessed weekly. Preliminary experiments showed that amnion-derivedcells sometimes form small floating clusters in suspension cultureconditions. These clusters must be dispersed to ensure accurate cellcounts and this was achieved by incubating the cultures in trypsin for5-10 minutes prior to counting. Cells were counted, and viabilityassessed, using a Guava PCA-96 Personal Cell Analyzer (ViaCountpackage). As little as 20 μl of sample was used for this analysis. Agraph of total and viable cell counts per ml was plotted with time toensure that the cells were able to divide and remain viable over time inculture. Cells were subcultured at 1.3−1.5×10⁶ cells per well. Thecultures were maintained until proliferation ceased. Control adherentcultures were maintained on tissue culture treated 6-well plates tocompare rates of proliferation between adherent and suspension culture.Cultures were maintained at 37° C., in 5% CO₂ in air. Passage ofadherent cells was performed at confluency. Adherent cultures weretrypsinized and washed before replating at 1.3×10⁶ cells/well. Cellcounts and viability were measured in the adherent cells at eachpassage. Culture media was tested on 5 different donor tissues toaccount for tissue variability.

Results: Of the 5 placentas tested, 4 showed at least 2 foldproliferation at least through day 20 under all conditions tested, thusdemonstrating that non-adherent static culture methods are a feasiblealternative to microcarrier bead or adherent flask culture.

Example 4 Addition of Growth Factor Additives to Promote More ExtensiveProliferation

After selection of a culture medium that supports suspension culture in6-well plates, various growth factor additives are tested to promotemore extensive proliferation in suspension culture conditions. Thesegrowth factors include EGF, IGF-1, IGF-II, αFGF, αFGF-h, βFGF, FGF-4,FGF-8, KGF, SCF, Fsk, SHh, Prog, Wnt-1, CT, VPA. These and other factorsknown to either promote cell proliferation or decrease apoptosis oranoikis are tested at various concentrations in the suspension culturesto determine their effect on proliferation. Additional testing isperformed to ensure that these factors are not promoting differentiationor changing the secretory profile of the cells.

Example 5 Culturing of Amnion-Derived Cells in Spinner Flasks or RollerBottles without Microcarrier Attachment

After selection of a culture medium that supports proliferation ofamnion-derived cells in suspension, with or without the addition ofgrowth factors other than EGF, experiments are performed to assess theproliferation of the cells in a stirred bioreactor. Amnion-derived cellsare placed into the spinner flasks (suspension cells; 3×10⁵ cells/ml).Adherent cells cultured in T-flasks are used as controls (adherentcells; 1.3×10⁵ cells/cm²). All cultures are incubated at 37° C. in 5%CO₂ in air. The spinner flasks are treated with Sigmacote(Sigma-Aldrich) prior to use, to prevent the cells from attaching to theglass. The flasks are agitated by placing them on magnetic stir-plate.Daily samples of cells are withdrawn, in a class II biosafety cabinet,from each spinner flask, and the number of cells is counted, andviability assessed, using a Guava PCA-96 Personal Cell Analyzer(ViaCount package). The use of multiple spinner flasks permits the useof 2 or 3 replicates of each condition per experiment.

One of the challenges of a spinner flask system is the exposure of thecells to sheer forces caused by the rotating impeller. A gentleralternative to spinner flasksis to culture the cells in roller bottles.Tissue culture treated roller bottles (1.2 L; Corning) are pre-treatedwith poly 2-hydroxyethyl methacrylate (Poly-Hema) to prevent cellattachment. Roller bottles containing 30−40×10⁶ amnion-derived cells areplaced on a roller bottle apparatus (Integra Biosciences). Cells aresampled on a biweekly basis and assessed for viability andproliferation, as indicated above.

Example 6 Generation of Monoclonal Antibodies

In one embodiment, Balb/c mice are immunized with amnion-derived cellspreviously cultured for up to 5 days, preferably 1-2 days. In thisembodiment, both adherent and non-adherent cells are recovered from theculture and used to immunize the mice. In another embodiment, afterculturing for up to 5 days, preferably 1-2 days, only the adherent cellsare recovered and used for immunization of the mice. In anotherembodiment, after culturing for up to 5 days, preferably 1-2 days, onlythe non-adherent cells are recovered and used for immunization of themice. Four to six weeks after immunization, the spleens are removed andthe spleen cells are fused to a mouse myeloma cell line, SP2/0-Ag14,using techniques known in the art, resulting in the generation of viablehybridoma cells. As many as 1000 hybridomas may be expanded, screenedfor the expression of monoclonal antibodies, and further tested fortheir specific reactivity to cell-surface protein markers onamnion-derived cells. Antibody samples are analyzed by flow cytometryand, along with commercially available antibodies, will be used toidentify unique protein markers on amnion-derived cells. Thus, theinvention is directed to hybridomas producing the monoclonal antibodiesof the present invention as well as the monoclonal antibodies.

Example 7 Antibodies which React with Amnion-Derived Cell SurfaceProtein Markers

Monoclonal antibodies which react with amnion-derived cell proteinmarkers on the surface of amnion-derived cells may be used to separatethe cell population into substantially purified population ofamnion-derived cells and will be useful in characterizing eachsubstantially purified population of amnion-derived cells for its stemcell characteristics. The monoclonal antibodies of the present inventionmay be used to isolate a stem cell protein marker unique to thesubstantially purified population of amnion-derived cells. The newlyidentified protein marker may be used to isolate a nucleic acid sequenceencoding the protein. Thus, the invention is directed to unique markerson amnion-derived cells, the isolated protein marker, the isolatednucleic acid encoding the protein marker, as well as expression vectorscapable of expression the protein marker when transfected into mammaliancells such as CHO, COS, etc. The invention is further directed tobacterial cells carrying the vector for vector propagation.

In addition, the invention contemplates using known antibodies toidentify and create substantially purified populations of amnion-derivedcells having unique combinations of markers useful as identifyingcharacteristics of the substantially purified populations. This uniquecombination of markers can be used to isolate, characterize, purify orcreate a substantially purified population of amnion-derived cellshaving those characteristics.

All cell characterizations described herein were done using freshlyisolated amnion-derived cells. One of skill in the art will recognizethat the expression pattern of the markers may vary depending uponculture conditions and time in culture. For example, the protein markerexpression pattern seen in the expanded populations of the inventiondescribed herein may be different from that seen in freshly isolatedamnion-derived cells. In addition, one of skill in the art willrecognize that the order in which the cells are contacted with theantibodies is not critical to obtaining the desired populations ofamnion-derived cells. Table 2 shows the results of FACS analysis ofamnion-derived cells freshly isolated from the amnion of a placenta. Theantibodies used, alone or in combination, may be useful to identify,isolate, characterize or create a substantially purified population ofamnion-derived cells. One preferred embodiment is the use of anti-CD90and anti-CD 117 antibodies to identify, isolate, characterize or createa substantially purified population of amnion-derived cells. Otherpreferred embodiments for identifying, isolating, characterizing orcreating substantially purified populations of amnion-derived cellsinclude contacting the cells with anti-CD90, anti-CD117, and anti-CD105antibodies; contacting the cells with (i) anti-CD90, anti-CD117, andanti-CD105 antibodies and (ii) with at least one antibody selected fromthe group consisting of anti-CD140b, anti-CD34, anti-CD44, and anti-CD45antibodies; contacting the cells with (i) anti-CD90 and anti-CD117antibodies and (ii) with an anti-CD29 antibody; contacting the cellswith (i) anti-CD90, anti-CD117 and anti-CD105 antibodies and (ii) withan anti-CD29 antibody; contacting the cells with (i) anti-CD90,anti-CD117 antibodies and (ii) anti-CD29 antibodies and (iii) with oneor more antibodies selected from the group consisting of anti-CD9,anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R,anti-SSEA-4, and anti-HLA-G antibodies; contacting the cells with (i)anti-CD90, anti-CD117, and anti-CD105 antibodies and (ii) and anti-CD29antibodies and (iii) with one or more antibodies selected from the groupconsisting of anti-CD9, anti-CD10, anti-CD26, anti-CD71, anti-CD166,anti-CD227, anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies;contacting the cells with (i) anti-CD90, anti-CD117, and anti-CD105antibodies and (ii) and anti-CD29 antibodies and (iii) with one or moreantibodies selected from the group consisting of anti-CD140b, anti-CD34,anti-CD44, and anti-CD45 antibodies; b) contacting the cells with (i)anti-CD90, anti-CD117, and anti-CD105 antibodies and (ii) and anti-CD29antibodies and (iii) one or more antibodies selected from the groupconsisting of anti-CD140b, anti-CD34, anti-CD44, and anti-CD45antibodies and (iv) one or more antibodies selected from the groupconsisting of anti-CD9, anti-CD10, anti-CD26, anti-CD71, anti-CD166,anti-CD227, anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies; andcontacting the cells with one or more antibodies selected from the groupconsisting of anti-CD90, anti-CD117, anti-CD140b, anti-CD34, anti-CD44,and anti-CD45 antibodies; and one or more antibodies selected from thegroup consisting of anti-CD29, anti-CD9, anti-CD10, anti-CD26,anti-CD71, anti-CD166, anti-CD227, anti-EGF-R, anti-SSEA-4, andanti-HLA-G antibodies.

TABLE 2 Population % Designation Surface Marker ~95-100% +++ CD9 CD29~70-95% ++ SSEA4 CD10 CD166 CD227 ~60-95% + HLA-G EGFR CD26 ~10-50% +/−CD71 <1% − CD34 CD44 CD45 CD140b CD90 CD105 CD117

Table 3 shows where antibodies useful for practicing the methods of theinvention can be obtained.

TABLE 3 Antibody Antibody Name Manu. Cat. # Name Manu. Cat. # CD117 PEBD-Pharm 555714 CD44 APC BD-Pharm 559942 CD44 PE BD-Pharm 555479 CD45APC BD-Pharm 555485 CD45 PE BD-Pharm 555483 CD140b PE BD-Pharm 558821EGFR PE BD-Pharm 555997 CD90 Biotin BD-Pharm 555594 CD105 FITC ChemiconCBL418F CD26 BD-Pharm CD117 APC BD-Pharm 550412 CD166 BD-Pharm CD29 APCBD-Pharm 559883 CD10 BD-Pharm CD34 APC BD-Pharm 555824 CD71 BD-PharmCD227 BD-Pharm CD9 BD-Pharm

Example 8 Generation of Enriched Populations of Amnion-Derived Cells

Amnion-derived cell protein markers expressed on the cell surface may beused to enrich for populations of amnion-derived cells expressing thoseprotein markers using a variety of methods. Such procedures may involvea positive selection, such as passage of sample cells over a columncontaining anti-protein marker antibodies or by binding of cells tomagnetic bead-conjugated antibodies to the protein markers or by panningon plates coated with protein marker antibodies and collecting the boundcells. Alternatively, a single-cell suspension may be exposed to one ormore fluorescent-labeled antibodies that immuno-specifically bind toamnion-derived cell protein markers. Following incubation with theappropriate antibody or antibodies, the amnion-derived cells are rinsedin buffer to remove any unbound antibody. Amnion-derived cellsexpressing the protein marker(s) can then be sorted byfluorescence-activated cell sorting (FACS) using, for example, a BectonDickinson FACStar flow cytometer. To enrich for populations of cellsexpressing a desired protein marker(s), the cells may be subjected tomultiple rounds of FACS sorting.

In addition, protein markers that are not expressed on the surface ofamnion-derived cells may also be used to enrich for populations ofamnion-derived cells not expressing those markers. Such procedures mayinvolve a negative selection method, such as passage of sample cellsover a column containing anti-protein marker antibodies or by binding ofcells to magnetic bead-conjugated antibodies to the protein markers orby panning on plates coated with protein marker antibodies andcollecting the unbound cells. Alternatively, a single-cell suspensionmay be exposed to one or more fluorescent-labeled antibodies thatimmuno-specifically bind to the protein markers. Following incubationwith the appropriate antibody or antibodies, the cells are rinsed inbuffer to remove any unbound antibody. Cells expressing the proteinmarker(s) can then be sorted by fluorescence-activated cell sorting(FACS) using, for example, a Becton Dickinson FACStar flow cytometer andthese cells can be removed. Remaining cells that do not bind to theantibodies can then be collected. To enrich for populations of cellsthat do not express a desired protein marker(s), the cells may besubjected to multiple rounds of FACS sorting as described above.

Non-limiting examples of antibodies that may be useful to generate suchenriched populations of amnion-derived cells include anti-CD10,anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R, anti-SSEA4,and anti-HLA-G antibodies.

Alternatively, antibodies may be useful to generate enriched populationsof amnion-derived cells by removing undesired cells (i.e. by conjugatingantibodies to beads and adding the beads to a culture dish containing aheterogeneous population of amnion-derived cells such that cells in theheterogeneous population that express the marker to which the antibodyis directed will bind to the beads thus removing them from thepopulation of cells that do not express the marker). Non-limitingexamples of antibodies that may be useful in this process anti-CD140b,anti-CD34, anti-CD44, and anti-CD45, anti-CD90, anti-CD105, andanti-CD117 antibodies.

Example 9 Monoclonal Antibody Library

To construct a “monoclonal antibody library” a collection of severalmonoclonal antibodies may be selected which identifies and isolates theparticular cell population responsible for the multipotent cell activitycharacteristic of the population of amnion-derived cells of the presentinvention. The panel of monoclonal antibodies may be reacted withplacental tissue, a placental-derived cell suspension, or a culture ofplacental-derived cells. Cells reacting with the collection ofmonoclonal antibodies may be identified and isolated using methods knownin the art, e.g., FACS. The invention is therefore directed to acollection of monoclonal antibodies that are used to form a monoclonalantibody library.

Example 10 Use of Amnion-Derived Cell Compositions in Wound Healing

Methods. The keratinocyte cell line isolated from epidermis (ATCCCRL-1555) was seeded onto 6-well plates at a density of 0.3×10⁶ cellsper well. Cells were left to grow to confluency then placed intoserum-free conditions for 48 hours. In each well a scrape or wound ofthe confluent monolayer was made from the top to the bottom of the wellusing a 1 ml pipette tip. Images of the scrape were taken at 0, 24, 30and 48 hours to determine cell migration or percent of wound closure inresponse to addition of conditioned medium to each well. Conditionstested were 0%, 50%, and 100% of the following: 1) No conditioned media(control, 0%); 2) Conditioned media from amnion-derived cells passagednormally at ratio of 1:3; 3) Conditioned media from amnion-derived cellsthat were never passaged; 4) Conditioned media from amnion-derived cellsgrown in the ATCC cells' media; and 5) Conditioned media from ATCC cellsgrown in their own media. Approximately 6 measurements were taken inmicrons of each scrape at each time point using phase microscopy andMetaMorph imaging software. The percent of healing was calculated bycomparing the width of each wound at 24, 30, and 48 hours to thestarting width of the wound at time zero.

Results. Conditioned media (CM) from amnion-derived cells showed asignificant increase in cell migration or healing of the scrape comparedto control. CM from other cell types, however, did not show thisincrease. Cells that grew in CM from amnion-derived cells were the onlycondition that showed complete closure of the scrape before 24 hours. CMfrom cells passaged at a ratio of 1:3 and at a concentration of 50%(CM/non-CM) produced the best results. These results suggest thatcomponents of CM from amnion-derived cells have properties that increasecell migration or wound healing.

Example 11 Amnion-Derived Cells, Conditioned Media, and Cell LysatesAccelerate Re-epithelialization, Collagen Synthesis, and Regain toTissue Tensile Strength

The following experiment was done to assess whether the application ofamnion-derived cells, amnion-derived cell conditioned media oramnion-derived cell lysates could: 1) accelerate the rate ofre-epithelialization, 2) accelerate collagen synthesis and deposition inthe wound bed and 3) speed up regain to tissue tensile strength anddemonstrate that transplantation of stem cells may have the sameproperties. It was also done to assess whether transplantedamnion-derived cells could incorporate into epidermal and dermalstructures including follicles, glands and blood vessels.

Animal model: This initial study utilized a total of 90 rats,distributed into the following groups (5 sacrifice time points, 3animals per treatment group, 6 groups per time point), Table 4.

TABLE 4 Time Points (days) Group # Treatment 3 5 7 14 21 1 Control (notreatment) 3 3 3 3 3 2 Non-conditioned media + gelfoam 3 3 3 3 3 3Amnion-derived cell conditioned 3 3 3 3 3 media + gelfoam 4 Hyaluronicacid vehicle 3 3 3 3 3 5 Amnion-derived cell + hyaluronic 3 3 3 3 3 acidvehicle 6 Amnion-derived cell lysate + hyaluronic 3 3 3 3 3 acid vehicle

Each animal received 2 dorsal, full-thickness excisional wounds, for atotal of 180 wounds for the entire study, with 6 wounds/group/timepoint.

Skin wounding: A pair of wounds was made on each side of the dorsalmidline, using a disposable punch biopsy (6 mm diameter) These woundswere full-thickness through the epidermis and dermis. Wounds weretreated with: nothing (control), vehicle (10 mm Gelfoam sponge saturatedwith non-conditioned media), conditioned media (10 mm Gelfoam spongesaturated with amnion-derived cell conditioned media), hyaluronic acidvehicle (0.1 ml of Hylan A gel, Genzyme Corporation), hyaluronicacid+fluorescently (CM-DiI dye, Molecular Probes, Eugene Oreg.) labeledamnion-derived cells (10⁶ cells/wound) or hyaluronic acid+amnion-derivedcell lysate (from 10⁶ cells/wound), immediately following injury (Seeabove Table 5). The entire dorsal skin was covered with a steriledressing (Tegaderm, 3M, Minneapolis, Minn.) secured with a biocompatibleadhesive (Mastisol, Ferndale Laboratories Inc, Ferndale, Mich.). Woundsin the first three treatment groups were re-treated in an identicalmanner on days 2, 3, 4 and 5 post wounding. Following the 5th woundtreatment, the wounds were left undisturbed until day 7, at which timethe Gelfoam as well as the sterile dressing was removed and the woundallowed to heal exposed to the surrounding environment. Wounds in thelast three treatment groups were left undisturbed until time ofsacrifice.

Imaging and clinical assessment: Two blinded observers assessed thedegree of wound healing for each of the 180 wound samples at thefollowing days post injury: 1, 2, 3, 4, 5, 7, 14 and 21. The followingparameters were ascertained: hemostasis, wound contraction,re-epithelialization and inflammation. Digital images were taken ofrepresentative wound samples for each treatment group and stored forlater analysis.

Tissue analysis: Animals were be euthanized according to the above timetable by intracardiac administration of pentobarbital sodium andphenyloin sodium following heavy sedation with ketamine/xylazine. Dorsalskin was removed using aseptic technique and each wound was individuallydissected and divided. One half of each wound was used for tensilestrength measurements, with the other embedded for frozen sectioning andimage analysis.

Tensiometry: Wound samples from the day 7, 14 and 21 groups wereanalyzed by tensiometry. For tensile strength measurements the frozenspecimens were trimmed of sub-cutaneous fat and any muscle that wastaken along with the biopsy, and divided into 4-5 samples. Thecross-sectional area of each specimen was measured with calipers. Thenthe specimen was clamped in the tensiometer, and force exerted until theskin teared. Measurements were recorded by a computer and tensilestrength calculated using the formula: Maximum Tensiometer Reading(converted to g) divided by Cross-sectional Area (sq−mm)=Tensilestrength (g/sq−mm). The results for individual specimens from one woundwere combined to determine an average TS/wound (tensile strength perwound). This value was normalized for the TS/skin (tensile strength ofuninjured skin from the opposite side); TS/wound divided byTS/skin=relative TS/wound. The relative TS/wound was tabulated for eachgroup at each time point and the mean and standard deviations determinedusing Excel database software (Microsoft Office 2000).

Microscopic analysis: Tissue specimens were embedded in O.C.T. (Miles,Inc., Elkhart, Ind.) and cryostat-sectioned into approximately 10 μmthick sections, at −23° C. Thin sections, mounted on glass microscopeslides, were stored in moisture-proof slide boxes at −70° C.Representative slides were processed for immunohistochemicalcharacterization of the connective tissue components using standardtechniques. Hematoxylin and eosin staining were used to ascertain theoverall histological appearance of the injured mucosa. Collagen presencein the wound was assayed using Masson's trichrome stain.Picrosirius-polarization method was used to analyze collagen fiberorganization. Grafting and survival of fluorescently labeled stem cellsin the wound bed was semi-quantitatively analyzed by measuring the totalamount of fluorescence present in the wound bed. Localization of cellswas recorded and analyzed.

Effect on the rate of wound re-epithelialization and dermal collagendeposition and organization was determined. Each of these, as well asother components of the wound healing process, were analyzed usingspecific markers. Transplantation of live amnion-derived cells into thedermal wound bed was expected to result in: 1) differentiation andengrafting of stem cells into various skin compartments and 2) continualregulated release of various stem cell factors.

Results—The results of this experiment are set forth in Table 5 below.

TABLE 5 Amnion-derived cell Summary conditioned medium Amnion-derivedcells Positive 1. Day 5: CM wounds, 1. Re-EpithelializationEffects >contracted granulation in early time points formation 2.Angiogenesis in early 2. Day 14: CM wounds appeared time pointssmaller, >contracted, >healed 3. Dynamics of collagen 3. CM woundsexhibited faster re- deposition and epithelialization and angiogenesis.organization Negative None No detection of engrafted Effects cellsUnaltered 1. Synthesis and deposition 1. Clinical observations ofcollagen 2. Regain of tissue 2. Regain of tissue tensile tensilestrength strength

As shown in Table 5, treatment of wounds with amnion-derived cellconditioned media showed an increase in contracted granulation formationby Day 5, and smaller wounds, greater contraction and healing by Day 14.In addition, the wounds exhibited faster re-epithelialization andangiogenesis as compared to controls. Synthesis and deposition ofcollagen and regain of tissue tensile strength were unaltered over thecourse of the experiment. Treatment of wounds with amnion-derived cellsshowed re-epithelialization and angiogenesis at early time points, aswell as evidence of collagen deposition and organization. Engraftedcells were not detected. No differences based on visual inspection inclinical observations (redness, swelling, size, etc.) were seen nor wasregain of tissue tensile strength altered over the course of theexperiment.

Example 12 Detection of Cytokines in Conditioned and Unconditioned MediaSamples

In addition to pluripotency, amnion-derived cells may play a significantrole in the inflammatory response. In the early phases of wound healing,chemokines and cytokines regulate chemotaxis and activation ofinflammatory cells. Growth factors play dominant roles in regulatingcell proliferation, differentiation, and synthesis of extracellularmatrix. Amnion epithelial cells have been shown to secrete manycytokines and growth factors. These factors include prostaglandin E,PDGF, TGF-α, EGF, IL-4, IL-8, TNF, interferons, activin A, noggin,b-FGF, angiogenic factors, and other neuroprotective factors (Koyano,S., et al., (2002) Dev Growth Differ 44, 103-12; Blumenstein, M., etal., (2000) Placenta 21, 210-7; Tahara, M., et al., (1995) J ClinEndocrinol Metab 80, 138-46; Paradowska, E., et al., (1997) Placenta 18,441-6; Denison, F. C., et al., (1998) Hum Reprod 13, 3560-5; Keelan, J.A., (1998) Placenta 19, 429-34; Sun, K., et al., (2003) J ClinEndocrinol Metab 88, 5564-71; Uchida, S., et al., (2000) J Neurosci Res62, 585-90).

Many of these cytokines are associated with wound healing and some havebeen credited with contributing to scarless healing in the fetus(Robson, M. C., et al., (2001) Curr Probl Surg 38, 72-140; Ferguson, M.W. et al., (2004). Philos Trans R Soc Lond B Biol Sci 359, 839-50).

To determine which of these cytokines may be secreted by theamnion-derived cells of the present invention, conditioned media fromamnion-derived cells was isolated from cell cultures that were seededonto tissue culture treated flasks at a density of ˜40,000 cells percm². Cells were cultured in a proprietary serum-free medium supplementedwith 10 ng/ml of EGF. Culture media was exchanged every 2 days duringthe growth period. After cells reached near confluency (˜1-2 wk afterisolation), fresh media was applied and conditioned media was collectedafter three days and stored at −80 C for subsequent analysis.

Conditioned media was analyzed for secreted protein content via antibodyarrays for multiple protein detection (RayBiotech, Norcross, Ga. usingRayBio® Human Cytokine Antibody Arrays V, VI, and VII). The samples thatwere analyzed are shown in Table 6 below.

TABLE 6 1. Complete unconditioned media + plasbumin 2. Completeunconditioned media + EGF (no plasbumin) 3. Conditioned media fromplacenta 1 + plasbumin 4. Conditioned media from placenta 1 (noplasbumin) 5. Conditioned media from placenta 2 + plasbumin

Results—Table 7 provides the results of this experiment.

TABLE 7 Wound Healing Relevant Cytokines Positive in Conditioned MediaNegative Angiopoietin-2, Angiogenin, bFGF, EGF, FGF-7, TGF-a, TGF-beta1, FGF-4, IGF-1, IL-1 beta, IL-2, IL-4, IL-6, IL-8, TGF-beta 2, TGF-IL-10, PDGF-AA, PDGF-AB, PDGF-BB, PDGF- beta 3 Ra, PDGF-Rb

Example 13 Amnion-Derived Cell/Fibroblast Co-Cultures

It has been reported in the literature that under certain conditionswhen ES cells are co-cultured with fibroblasts, the ES cells are inducedto differentiate into keratinocyte-like cells. To determine what effectco-culture of amnion-derived cells with fibroblasts would have onamnion-derived cells, an experiment was done in which 3.3×10̂6amnion-derived cells were co-cultured with 0.4×10̂6 fibroblasts on acollagen IV-coated T25 flask for 3, 5, 10, 15, and 25 days.

Results—When treated with the trypsin-like enzyme Tryple (Invitrogen),both amnion-derived cell cultures and fibroblast cell cultures alonerelease cells as a single cell suspension. However, when theamnion-derived cell/fibroblast co-culture was treated with Tryple, thecells came off the treated culture surface as sheets rather than as asingle cell suspension. Furthermore, the sheets were very stable andsomewhat resistant to enzymatic and mechanical disruption.

It is theorized that these sheets may be suitable for use as wounddressings when it is desirable to have a dermal-type graft. Withdemonstrated recent success with mitral resuscitation, management ofinhalation injuries, control of burn wound sepsis, and understanding ofthe hypermetabolic response, early excision and rapid closure of theburn wound with a serviceable integument becomes a therapeuticimperative. In small surface area burns, this can be accomplished byautogenous skin grafts. For large surface area burns, both partial andfull-thickness, there is not yet a totally satisfactory solution.Cutaneous epithelial autografts can be grown from the patient's skin andmassively expanded to cover the entire body. Unfortunately, the lack ofdermis leads to prolonged fragility and significant scarring, therefore,many believe that a “dermis” is required along with an epithelium.

Recent products with a supposed dermal substitute or neodermis such asIntegra, Alloderm, Transcyte, Apligraf, and Dermagraft have attemptedsolve the problem. However, all of these “skin” substitutes have theproblem of being expensive and having lower resistance to infection thanautografts. Without a satisfactory rapid reliable wound closure for burninjuries, the wound remains in the inflammatory phase of healing for aprolonged period of time resulting in excessive scarring.

Robson et. al., (Robson, M. C., and Krizek, T. J. (1973) Ann Surg 177,144-9.) reported success in treatment of experimental and clinical burns(both partial and full thickness) using human amniotic membranes. It wasthought that part of the effect seen from the treatment with amnioticmembranes was due to a humoral substance or substances stimulating woundhealing. These observations were prior to present knowledge of cytokinesand growth factors. More recently, attempts have been made to userecombinant growth factors and growth hormones to affect more rapidhealing of the burn wound. Amniotic membranes proved not to be practicalbecause of the risk of virally transmitted diseases. However, theobservations from those early experiments and coupled with new knowledgesupport the possibility that the pluripotentiality of amnion-derivedcells and their now demonstrated protein secretory profile of cytokinesand other humoral substances stimulatory for wound healing may be usefulin providing rapid early closure for thermal injuries.

Example 14 Effects of Amnion-Derived Cell Conditioned Media in an AnimalModel of Acute Wound Healing

An animal model of acute excisional granulating wound was used toevaluate the effect of amnion-derived cell conditioned media on woundhealing.

Methods: Acute excisional granulating wound model: Twenty maleSprague-Dawley rats weighing 250-300 g were anesthetized using ketamine(40 mg/kg), xylazine (10 mg/kg) and acepromazine (0.75 mg/kg). Followinganesthesia, the dorsum of each animal was depilitated and foursymmetrical midline areas 1.5×1.5 cm were traced on the skin using acopper template. Four wounds were then created by excision of the markedareas through the skin and the panniculus carnonsus muscle. The animalswere divided into the following groups of 5 (Table 8):

TABLE 8 Group No. Experimental Conditions I Conditioned media,non-infected II Unconditioned media, non-infected III Conditioned media,infected IV Unconditioned media, infected

Analog tracings were made every 72 hours onto acetate sheets of bothopen wound areas and of the advancing full-thickness skin edges of allwounds. To eliminate site-related variability in the wounds, only thethree caudal wounds were measured for statistical purposes, since themost cephalad wound has been shown to demonstrate different healingcharacteristics. Wound area calculations were performed with the use ofdigital planimetry (Sigma Scan; Jandel Scientific, Corte Modera,Calif.). Weekly quantitative bacterial analyses were performed on asubset of wounds in each group and are expressed as CFUs/g of tissue.

After all four wounds of each animal were completely epithelialized asdetermined by visual inspection, the animals were euthanized and theentire dorsum of the rat including the panniculus carnosus was removed.A 1 cm wide skin strip perpendicular to each resultant scar, washarvested for breaking strength analysis. An Instron tensiometer (ModelNo. 4201; Instron Corp., Canton, Mass.) with a 5 kg tension load celland cross head speed of 10 mm/min was used. Breaking strength is definedas the force required to rupture the scar and is reported in kilograms.

Results—The application of conditioned media overcomes the inhibition ofwound healing caused by bacteria and shifts the healing trajectory incontaminated wounds to that of near normal healing (FIG. 2).

Example 15 Effects of Amnion-Derived Cell Conditioned Media in an AnimalModel of Chronic Wound Healing

Methods: Chronic granulating wound model: Twenty male Sprague-Dawleyrats weighing 300-350 g are anesthetized using ketamine (40 mg/kg),xylazine (10 mg/kg) and acepromazine (0.75 mg/kg). Following anesthesia,the dorsum of each animal is shaved and depilitated. A full-thicknessdorsal burn measuring 30 cm³ is created by immersion in boiling water.Animals in the contaminated group are seeded with 5×10̂8 Escherichia coliATCC #25922 after the rats have been allowed to cool for 15 minutes.Bacteria is obtained from fresh 18 hour broth culture and inoculum sizeis confirmed by backplating. The animals are divided into 8 equal groupsof 5 for different treatments after the day 5 escharectomies.

Animals are individually caged and given food and water ad libitum. Fivedays after burning, the eschar is excised from anesthetized animals,resulting in a chronic granulating wound. Histological characteristicsof this wound with comparison to human granulating wound have beenpreviously performed. The wounds are treated with the same experimentalgroups as described in Table 9 above. Any dried exudates that form areatraumatically removed prior to wound tracings or biopsies. Every 72hours the outlines of the wounds are traced onto acetate sheets and areacalculations are performed using digital planimetry. Care is taken onlyto record the advancing full-thickness margin rather than any advancingedge of epithelium. This avoids the small component of advancementprovided by the smooth, pink, translucent, hairless neoepithelium.Serial area measurements are plotted against time. For each animal'sdata, a Gompertz equation is fitted (typical r2=0.85). Using this curvethe wound half-life is estimated. Comparison between groups is performedusing life table analysis and the Wilcoxon rank test. The statisticalanalysis is done using the SAS (SAS/STAT Guide for Personal Computers,Version 6 Edition, Cary, N.C., 1987, p. 1028).

Example 16 Effects of Amnion-Derived Cells in Two Animal Models of WoundHealing

The two animal models of granulating wounds described above in Examples14 and 15 are used to evaluate the effect of amnion-derived cells onwound healing. The experimental groups are as follows in Table 9.

TABLE 9 Group No. Experimental Conditions I Non-contaminated control(PBS only) II Contaminated control (PBS + bacteria) III Non-contaminatedtreated with cells IV Contaminated treated with cells

Example 17 Ability of Amnion-Derived Cells to Promote CompleteRegeneration of Deep Wounds

Experiments are designed to promote complete regeneration of deep woundsthrough re-creating the all of the necessary tissues including bone,muscle, cartilage, skin, and neural tissue. Initially, in vitroexperiments are designed to determine if amnion-derived cells candifferentiate into all of the cells of interest. Amnion-derived cellswill be cultured as previously described. Mesenchymal stem cells(Cambrex, Rutherford, N.J.) will be used as a control fordifferentiation experiments. MSC's will be seeded at 5,000-6,000 cellsper cm² and cultured in Mesenchymal Stem Cell Growth Medium (MSGM,Cambrex, Rutherford, N.J.).

Osteogenic: Once cells are confluent, growth media will be changed(DMEM, 10% FBS, 1% pen/strep) to osteogenic differentiation media (Shi,Y. Y., et al., (2005) Plast Reconstr Surg 116, 1686-96.) (DMEM, 10% FBS,1% pen/strep, 250 uM ascorbate-2-phosphate, 10 mM beta-glycerophosphate,2.5 uM retinoic acid). Osteogenic differentiation media will be changedevery 2-3 days. Alkaline phosphatase activity of adipose-derivedmesenchymal cells will be evaluated in duplicate wells after 7 days ofculture. Alkaline phosphatase staining will be performed using theAlkaline Phosphatase Staining Kit (Sigma) following the manufacturer'srecommendations. Experiments will be performed in triplicate. Von Kossastaining will be performed in duplicate wells to assess the ability ofcells to mineralize the extracellular matrix and form bone nodules.Staining will be performed on cells after 21 days of culture induplicate wells in differentiation media conditions. Cells will be fixedin neutral buffered formalin for 30 minutes, incubated with 1% aqueoussilver nitrate for 15 minutes under ultraviolet light, stained with 5%sodium thiosulfate for 2 minutes, and finally counterstained with 1%Safranin O for 10 minutes. In addition, calcium concentration in theextracellular matrix will be determined via a biochemical colorimetricassay using the Calcium Reagent Set (Biotron Diagnostics, Hemet, Calif.)in duplicate wells. Experiments will be performed in triplicate.

Adipogenic: Amnion-derived cells and MSC will be cultured in adipogenicdifferentiation media (Shi, Y. Y., et al., (2005) Plast Reconstr Surg116, 1686-96.) for 3 days (DMEM, 10% FBS, 1% pen/strep, 10 ug/mlinsulin, 1 uM dexamethasone, 0.5 mM methylxanthine, 200 uMindomethacin), then change to adipocyte maintenance media for 2 moredays (DMEM, 10% FBS, 1% pen/strep, 1 ug/ml insulin). Oil Red 0 stainingwill be performed to assess for adipogenic differentiation in duplicatewells (as indicated by the presence of intracellular lipid-filleddroplets) after 5 days of culture in adipogenic media. Cells will befixed in 10% neutral buffered formalin for 30 minutes and then incubatedin 60% Oil Red 0 solution for 30 minutes at 37° C. Experiments will beperformed in triplicate.

Chondrogenic: Amnion-derived cells and MSC will be cultured in standardnon differentiation conditions and then collected and resuspended at1×10⁷ cells/ml concentration. Ten μl droplets will then be placed onto aculture dish and allowed to adhere to substratum at 37° C. for 2 hours.Then chondrogenic media (Malladi, P., et al., (2006) Am J Physiol CellPhysiol 290, C1139-46.) will be added carefully around cell aggregates(DMEM, 1% FBS, 1% pen/strep, 37.5 ug/ml ascorbate-2-phosphate, ITSpremix (BD Biosciences), 10 ng/ml TGF-B1 (Research Diagnostics, Inc.,Flanders, N.J.). Micromasses will be fixed in 4% paraformaldehyde with4% sucrose for 15 minutes, embedded with Optimal Cutting Temperature(O.C.T.) compound. Ten μm cryosections will be mounted on slides andstained by hematoxylin and eosin and alcain blue. Immunohistochemistrywill be performed as follows. Sections will be blocked at roomtemperature for 30 minutes and incubated with primary antibody at 4° C.overnight (anti-collagen II, Santa Cruz Biotechnology, Santa Cruz,Calif.). Followed by secondary antibody (Vector Labs, Burlingame,Calif.) incubation, 8 sections will be labeled with ABC reagent (VectorLabs, Burlingame, Calif.) for 10 minutes at room temperature. DAB(Vector Labs, Burlingame, Calif.) was applied to each section andhematoxylin will be used for counterstaining.

Skeletal myogenic: Amnion-derived cells and MSC will be cultured aspreviously described. Skeletal myogenic differentiation will be inducedby culturing cells in myogenic medium (Gang, E. J., et al., (2004) StemCells 22, 617-24.) (culture medium supplemented with 5% horse serum, 0.1μM dexamethasone, and 50 μM hydrocortisone) for up to 6 weeks. Myogenicdifferentiation will analyzed by FACS for MyoD1, myogenin, and myosinheavy chain (MyHC). For FACS, cells will be detached and stainedsequentially with primary antibodies (human-anti MyoD and anti-myogeninantibodies; Becton Dickinson) and FITC-conjugated secondary antibodies(FITC-rat anti-human IgG1; Becton Dickinson). Cells will be fixed with2% formaldehyde until analysis with FACS. For detection of anintracellular protein MyHC, cells were permeabilized with coldmethanol/PBS for 2 minutes at −20° C. before staining with primary mouseanti-myosin (fast, Sigma) and FITC-conjugated secondary antibody.

Example 18 Evaluation of Accelerated Wound Strength and Prevention ofAcute Wound Failure

One object of the invention is to reduce the incidence of surgical woundfailure and to optimize surgical wound outcomes by treating these acutewounds with conditioned growth media from amnion-derived cells. Thefocus is muscle, fascial and skin wound healing in vivo followingsurgical injury. Wound fibroblasts are isolated to measure the effect ofsoluble mediators derived from amnion-derived cells on repair fibroblastfunction in vitro.

Methods: Male, Sprague-Dawley rats are used for all experiments. Ventralabdominal wall hair is shaved and the field is cleansed with alcohol andsterile water. A 6 cm full-thickness skin incision is placed 2 cmlateral to the ventral midline and a rectangular skin flap 4 cm in widthis subsequently fashioned and raised through the avascular prefascialplane exposing the linea alba. In the Sham Operated rats, this skin flapis replaced and sutured using 4-0 Prolene. In the Experimental grouprats, a 5 cm isolated laparotomy incision is placed through the midlineof muscular layer of the abdominal wall (linea alba). The design of theventral abdominal wall skin flap model allows laparotomy healing tooccur isolated from the overlying skin wound. In the Mechanically IntactWound group, the laparotomy is repaired with a running, 3-0polypropylene suture using 0.3 cm suture bites and 0.5 cm progressbetween stitches. The suture is tied to itself at the end of the wound.Experience with this model predicts a 100% intact wound healing rate. Inthe Hernia Wound group, the laparotomy incision is left un-sutured. Inboth the Disrupted Wound and Hernia Wound models, the skin flap issutured in place, acting as a sling to prevent the evisceration of theabdominal organs. Mortality using these models has been found to be lessthan 1%. Following 30 minutes of recovery on a heated pad, the rats arereturned to individual cages. Food and water are provided ad libitum.All rats are observed daily and weighed weekly. Experience with thesemodels predicts that by post operative day 28, 100% of the Intact Woundrats heal the laparotomy incision, and 100% of the Disrupted Wound ratsgo on to form incisional hernias.

Five rats are used at each time point to generate 5 distinct fibroblastcell lines for each of the four laparotomy wound types. Necropsies areperformed 1, 7, 14, 28 and 60 days after operations. In the DisruptedWound group, Day 0 is re-defined at the time of wound disruption onpost-operative day 3. The entire ventral abdominal wall is excised fromeach euthanized rat, and the skin separated from the muscular layer. Theperitoneal and subcutaneous (ventral) surfaces of each abdominal wall iscarefully inspected for the presence of laparotomy wound disruption andherniation. An incisional hernia is defined as minimum of 2 mm ofmyo-fascial separation and/or obvious trans-abdominal wall herniation ofabdominal contents. Biopsies are taken perpendicular to the axis of thelinea alba. One biopsy from each rat is immediately snap frozen inliquid nitrogen for subsequent RNA isolation and real-time PCRmeasurement of collagen and integrin expression. A second adjacentbiopsy from each rat is immediately fixed in 10% buffered formalin forsubsequent paraffin fixation and immunohistological analysis of woundstructure, fibroblast morphology, inflammatory response, angiogenesisand extracellular matrix formation. The final wound biopsy is placed inPBS (see Methods below). Primary, first pass fibroblast cell culturesare used to measure fibroblast proliferation, collagen synthesis andfibroblast populated collagen matrix compaction in vivo and followingcontrolled patterns of mechanical strain in vitro.

Rapid gain in wound strength is tested in this model as follows: Animalsare randomly assigned into one of 12 Groups (n=10 per group). InExperimental Designs 1 and 2, each of the three animal models (Shamlaparotomy, Healing laparotomy and Hernia) are treated with fourexperimental conditions of amnion-derived cell conditioned mediacontaining the humoral products of amnion-derived cells. (No treatment,Control amnion-derived cell media (0% conditioned), 50% amnion-derivedcell CM and 100% amnion-derived cell CM). See Table 10 for ExperimentalGroups. 100 IU of media is delivered to the site of the laparotomymyofascial and skin incisions prior to wounding. This is defined assimple priming and establishes that this is a reliable and efficient wayto deliver liquid growth media to the site of surgical incisions with alarge experience in this model. Fibroblasts are isolated from healingwounds over time and assayed for the effect of amnion-derived cell mediaon proliferation and collagen matrix compaction in vitro.

TABLE 10 Table of Experimental Groups No Treatment Unconditioned media(0% CM) 50% CM 100% CM Sham Sham Sham Sham Wound Wound Wound WoundHernia Hernia Hernia Hernia

Myofascia (laparotomy) and skin incision tensile strength rats arerandomly selected and euthanized at serial timepoints followinglaparotomy with an overdose of Nembutal (100 mg/kg i.p.). The entireventral abdominal wall is excised and the skin separated from themusculofascial layer. The wound healing interface is closely examinedfor evidence of acute failure (dehiscence) or primary incisional herniaformation, defined as a fascial defect greater than 2 mm on or after POD7. Fascial and skin sutures are removed. Two myofascial and two skinstrips in the shape of the uppercase letter “I” are taken perpendicularto the wound healing interface from each abdominal wall. A cuttingtemplate is used to mark the abdominal wall in order to minimize sizevariability between specimens. The abdominal wall myofascia and skinstrips are labeled and stored in PBS until tensiometric mechanicalanalysis is performed. Biopsies are taken of the myofascial (laparotomy)and skin wounds and immediately snap frozen in liquid nitrogen forbiochemical analysis or fixed in formalin for histology.

Mechanical testing of the abdominal wall fascial and skin strips isperformed within 6 hours of necropsy. The sample width and thickness ismeasured with Digimatic calipers (Mitutoyo American Corp., Chicago,Ill.). The samples are each loaded in tension to failure, during whichtime the force-extension data are collected. Force extension curves aregenerated using an Instron Tensiometer (model 5542, Instron Corporation,Canton, Mass.) equipped with a 50 Newton static load cell set at acrosshead speed of 10 mm per minute. Samples are mounted into the loadframe using pneumatic graspers, preloaded to 0.1 Newtons, and the gaugelength measured between the grips. The load frame applies tensile loadsperpendicular to the suture repaired wounds until mechanical tissuedisruption occurs. The anatomic location of the wound failure is notedfor each specimen. Force and tissue deformation data are simultaneouslyrecorded and captured on a computer connected to the load frame via adigital interface card. Data analysis is performed using the Merlinmaterials testing software package (Instron Corporation, Canton, Mass.).

Data from the stretch loading is used to determine the followingclinically important biomechanical properties: Breaking strength—themaximum load (F_(max)) at mechanical failure (Newtons); Tensilestrength—the maximum stress developed in the specimen per unit area,calculated as F_(max)/cross sectional area (N/mm²); Toughness—the energyabsorbed by the specimen under tension, calculated as the entire areaunder the force-extension curve from the origin to mechanical rupture(Joules); Elongation—the increase in length of the tissue under a load,defined as the length of the specimen at mechanical disruption minus theoriginal length (mm); Stiffness—the slope of the linear elastic regionof the force-extension curve (N/mm).

Histological analyses of provisional matrix structure, fibroblastmigration, inflammatory response and wound angiogenesis is used tocompare the groups using H&E and trichrome staining. The density ofwound collagen formation is measured using antibodies specific for ratcollagen types I and III (Chemicon International, Inc., Temecula,Calif.). Cellular infiltration into the wounds at each time-point ismeasured as the mean cell number from three high-powered fields by ablinded observer using a microscope. In addition, histological specimensare digitized using a UMAX Astra 1200S scanner and analyzed using thecomputer software application Adobe PhotoShop version 5.0. Differencesin cellularity and intensity of collagen staining are compared using theStudents t test (SigmaStat, Jandel).

Samples are collected from laparotomy wounds or incisional hernias fromrats or humans as described previously and are placed in a sterile 50 mLconical tube (Corning, Corning N.Y.) in cooled PBS and placed on ice.Each sample is minced into small pieces and placed in a sterile 6 cmdiameter Petri dish (Falcon, Franklin Lakes N.J.) containing 0.1%collagenase in PBS for 45 minutes at room temperature. During this time,tissues and cells are triturated several times using a tissue culturepipette. The solution is poured into a sterile 50 mL conical tube andcentrifuged at 800 rpm for 6 minutes. The collagenase in PBS issuctioned off and the remaining cell and macerated tissue pellet isreconstituted in 15 mL complete growth medium consisting of low glucoseDMEM (GIBCO, Grand Island N.Y.) supplemented with 10% newborn calf serum(GIBCO, Grand Island N.Y.), 25 μg/mL gentamicin (GIBCO, Grand IslandN.Y.), and 0.375 μg/mL amphoteracin B (Sigma, St. Louis Mo.). Cells aretransferred into a sterile T75 flask (Corning, Corning N.Y.) and placedinto an incubator at 37° C. with 5% CO₂. Complete growth medium ischanged every two days as soon as cells reached 10-15% confluence with aminimum of 6 colonies visible using an inverted microscope with the 5×objective. Standard cytokeratin, alpha smooth muscle actin, vimentin andvan Willebrand factor staining is done to precisely characterize thecells as fibroblasts.

Once cells reach confluence, they are passaged 1:2. The medium isremoved and the cell layer is washed with 10 mL of HBSS (GIBCO, GrandIsland N.Y.). Cells are trypsinized with 10 mL of 0.05% trypsin with0.53 mM EDTA (GIBCO, Grand Island N.Y.) for 4-6 minutes at 37° C. Thetrypsin is inhibited using 10 mL of complete growth medium. Cells arepoured into a sterile 50 mL conical tube and centrifuged at 600 rpm for5 minutes. The supernatant is removed and the cell pellet is resuspendedin complete growth medium. Cells are divided into flasks to give a finalpassaging concentration of 1:2. Cells are trypsinized and centrifuged asabove and reconstituted in 4 mL of DMEM with 40% newborn calf serum.This solution is divided into four 2 mL cryovials (Corning, CorningN.Y.), and 1 mL of cooled 20% DMSO (Fischer, Fairlawn N.J.) in DMEM isadded to each vial. Vials are placed in a container with isopropylalcohol and cooled at 1° C./min in a −80° C. freezer. When completelyfrozen, they are transferred to liquid nitrogen for storage.

One cryovial is removed from liquid nitrogen and quickly thawed in warmethanol. The contents are placed in a 50 mL conical tube with 20 mL warmcomplete growth medium and centrifuged at 600 rpm for 5 minutes. Thesupernatant is removed and the cellular pellet is reconstituted in 15 mLwarm complete growth medium. The cells are plated in a T75 flask. A MITcolorimetric assay is used to access viability of the fibroblasts bymeasuring their mitochondrial activity.

In vitro protein matrices (FPCL's) fashioned with collagen, fibrin, andfibronectin are used. Extracellular matrix protein lattices are preparedas described by the manufacturer (Upstate Biotechnology, Lake Placid,N.Y.). The gels are incubated for 24 hours at 4° C. The fibroblasts arecounted and their cell number adjusted to 1×10⁵ cells/ml. One hundredthousand first passage-cultured fibroblasts are added to eachprefabricated 3.5 cm lattice. The lattices are incubated at 37° C. with5% CO₂ and the extent of gel contraction is measured every 24 hours for5 days. The gels are digitally imaged each day and contractionmeasurements calculated using Sigma Scan software (Jandel Scientific,Corte Madera, Calif.). Alternatively, FPCL's fabricated from rat tailcollagen is used for corroborative data. This assay runs from 30 minutesto several hours and allows determination of the response of the cellsto various functional inhibitors. Measurement of gel contraction areperformed overtime. Collagen gels are detached from petri dishes,treated with 2.5% FCS or 2u/ml thrombin, and the diameter of the gelmeasured at perpendicular axis at various times. One function that isevaluated is the role of MAP kinases on collagen contraction.

To further characterize the fascial fibroblasts to explain the effectsseen from the animal experiments, a series of tests are performed on thewound fascial and dermal fibroblast cultures. These include collagentypes I and II gene expression using quantitative RT-PCR on extractedRNA; measurement of tissue collagen levels of biopsies of the woundhealing interface using the Sircol collagen assay method (AcurateChemical and Scientific Corp., Westburg, N.Y.); measurement offibroblast, alpha-1 and beta-1 integrin expression to assure that anyreduction in FPCL contraction was not due to poor migration and reducedfibroblast function; and immunohistochemistry to evaluate alpha-smoothmuscle action, and Proliferating Cell Nuclear Antigen (PCNA) usingspecific monoclonal antibodies for PCNA and alph-SMA (Sant CruzBiotechnology, Santa Cruz, Calif.).

Statistical analyses is as follows: For each experiment, a factorialdesign with balanced sample sizes in each group ensures that the maineffect, independent variables in each experiment can be isolated withappropriate statistical analysis. Outcome variables are compared usingparametric (continuous variables) and non-parametric (proportions)ANOVA. Nested ANOVA designs are used to incorporate the main effectvariables from each experiment into a single analysis. If the F ratiofor the overall ANOVA is significant, post hoc comparisons of individualgroup means are conducted via t-prime tests of least-squares means foreach comparison. Inherent in this approach is a Bonferonni correction ofthe significance level when making multiple pairwise comparisons. TheANOVA calculations are performed using the general linear models (GLM)algorithm from the Statistical Analysis System (SAS, Carey, N.C.), whichaccounts for unbalanced sample sizes, should they occur. Post-hocpairwise testing is conducted using the lsmeans option with the GLMprocedure. The correlations between the measured variables and ifsignificant covariance is observed between variables for a givenexperiment are examined, analysis of covariance (ANCOVA) is performed asappropriate. A 5% level of significance is considered statisticallysignificant.

Example 19 Use of Amnion-Derived Cells, Conditioned Media, Cell Lysates,and Cell Products for Rapid Early Wound Closure of Thermal Injuries

Outcome and rehabilitation of thermal injuries rely on early burn woundexcision and rapid wound closure. The speed of wound closure with aserviceable integument or integument substitute is the key to animprovement in survival. Providing novel approaches that will facilitateearly, rapid wound closure, while minimizing long-term scarring, is anobject of the present invention.

Established in vitro, animal models, and clinical patients are used toevaluate the use of amnion-derived cells for early, rapid wound closureof partial-thickness and full-thickness burns. In addition to thermalinjuries, experiments are done with established models for chemical,electrical, and cold injuries.

It is theorized that amnion-derived cells can differentiate intomesodermal and ectodermal cells. Thus, it may be possible that use ofsuch cells will provide early and permanent closure of the burn wound.Since presently, the prolonged time the wound is in the inflammatoryphase is the known variable leading to proliferative scarring, it isexpected that early, permanent closure of the burn wound would result indecreased scarring and, thus, increased function.

Methods: Three animal models of partial-thickness and full-thicknessthermal injuries are used. The three models are different because thefirst mimics partial-thickness healing by epithelialization inapproximately three weeks while the second and third mimicfull-thickness healing by contraction and epithelialization and canremain unhealed for up to eight weeks. The last two models have beenhistologically compared to the human granulating wound. The differencein the last two full-thickness wounds is the host. One group is a normalrat with an intact immune system, while the other is an athymic “nude”rat which is devoid of T-lymphocytes.

Partial-thickness burn injury: Forty female Hartley strain guinea pigsweighing 350 to 450 grams are used throughout this part of theexperiment. Under Nembutal anesthesia (35 mg/kg administeredintraperitoneally), the animals' backs are shaved and depilated. Auniform scald burn over 10% of the body surface is performed at 75° C.for 10 seconds. Guinea pigs are used for this model because of theirlack of an estrus (hair) cycle, and the ability to develop uniformpartial-thickness injuries in them. Animals are caged individually andfed food and water ad libitum.

At 24 hours, the animals are reanesthetized and the partial-thicknesseschar gently abraded. The 40 animals are divided into four groups of 10animals each. The groups are as follows: Group I —guinea pigs burned,abraded, and left untreated as controls; Group II— burned, abraded andtreated with nonconditioned media on day 1 (day of abrasion) and day 7;Group III— burned, abraded and treated with amnion-derived cellconditioned media on day 1 and day 7; Group IV— burned, abraded, andtreated with a suspension of amnion-derived cells over the entire burn,and dressed with Adaptic and bulky dressing. The outer dressing isgently removed every five days or prn. The animals are premedicated withbuprinorphine (0.1 mg/kg), anesthetized with halothane inhalation andburn wound biopsies are obtained on a weekly basis until the time ofhealing. The biopsy specimens are sectioned and stained, and the hairfollicles are counted microscopically and expressed as the number perhigh power field. Additionally, histological analyses of the healingskin is done. Gross observations are made and photographicallydocumented for the quality of healing and hair distribution.

Full-thickness burn injury (normal rat): Fifty male Sprague-Dawley ratsweighing 300-350 grams are acclimatized for one week prior to use. Underintraperitoneal Nembutal anesthesia, the rat dorsum is shaved anddepilated. A full-thickness dorsal burn measuring 30 square cms iscreated by immersion in actively boiling water. Seven mL of Ringer'slactate by subcutaneous injection is given to each rat to preventdehydration. Animals are individually caged and given food and water adlibitum. Five days after burning, the eschar is be excised fromanesthetized animals resulting in a granulating wound. Histologicalcharacterization of this wound with comparison to a human granulatingwound has previously been performed (Robson M C, et al., J Surg Res 16:299-306, 1974). The rats are divided into five groups of 10 animals eachand treated as follows: Group I receive no wound treatment and serve ascontrols; Group II receives treatment with nonconditioned media on day 0(day of escharectomy) and on day 7; Group III receives treatment withamnion-derived cell conditioned media on day 0 and day 7; Group IV istreated with a suspension of amnion-derived cells and dressed withAdaptic and a bulky dressing. The dressing is changed every five days orprn. Group V is treated with an extracellular matrix seeded withamnion-derived cells and dressed as in Group IV. Groups I-III animals'wounds are left exposed. Any dried exudates are atraumatically removedprior to any wound tracings or biopsies. Every 72 hours for rats inGroups I-III or whenever dressings require changing in Groups IV and Vthe rats are premedicated with buprinorphine (0.1 mg/kg), anesthetizedwith halothane inhalation, and the outlines of their wounds are tracedonto acetate sheets. Area calculations are performed using digitalplanimetry. Serial measurements are plotted against time. For eachanimal's data, a Gompertz equation is fitted (typical r2=0.85). Usingthis curve the wound half-life is estimated. Comparison between groupsis performed using life table analysis and the Wilcoxon rank test. Thestatistical analyses is performed using the SAS (SAS/STAT Guide forPersonal Computers, Version 6 Edition, Cary, N.C., 1987, p 1028) andBMDP (BMDP Statistical Software, Inc. 1988). From the best fit curvesfor the individual wounds, the number of days required for 25%, 50%, and75% healing of the original wounds is calculated. Randomized woundbiopsy sites are obtained from reanesthetized rats on days 5, 10, 15,20, and 25 post escharectomy (or days 10, 15, 20, 25, and 30 post burn)and placed in appropriate preservative solutions for histologicalstudies.

Full-thickness burn injury (immunologically impaired rat): Fiftyoutbred, congenitally athymic “nude” rats are purchased commercially(Harlan Sprague Dawley, Inc., Indianapolis, Ind.). All animals are maleand weigh between 250 and 300 grams. Because of their immune defect, theanimals are housed in pathogen-free barrier facilities, in cages withsealed air filters, animal isolators, laminar flow units, and laminarflow rooms. All supplies such as food, water, bedding etc. aresterilized to prevent infection. Procedures recommended in the Guide forthe Care and Use of the Nude Mouse in Biomedical Research (Institute ofLaboratory Animal Resources) are used at all times. Persons handling therats wear caps, masks, sterile gowns, sterile gloves, and shoe covers.All operations on “nude” rats are carried out under intraperitonealNembutal anesthesia, 35 mg/kg body weight, using aseptic surgicaltechniques. Operations are performed under a unidirectional airflowbiological hood. Surgical instruments are sterilized by autoclaving andsurgical sites are prepared with povidone iodine solution. The 50animals are divided into five groups of 10 each. The anesthesia,analgesia, procedures, wound treatments, and measurements are identicalto the intact rat model described above. Handling of the tracings,planimetry, and statistics are also the same as previously described.

In vitro fibroblast-populated collagen lattice: The fibroblasts areprepared as previously described by Kuhn, et al (Kuhn M A, et al.,Internat J Surg Invest 2: 467-474, 2001). The collagen lattices areprepared from type I rat tail collagen (acetic acid extracted) asrecommended by the manufacturer (Upstate Biotechnology, Lake Placid,N.Y.) (11). Undiluted collagen (1 ml) is placed in 35 mm culture dishes(Falcon 1008) and evenly spread. The dishes are placed in an ammoniavapor chamber for 3 minutes to solidify. Sterile distilled water (5 ml)is added to the culture dishes, allowed to stand for one hour, and thenaspirated. This is repeated four times to remove excess ammonia and thecollagen lattices are incubated for 24 hours at 4° C. PBS with 1.0%serum is added to replace the final aspirate. An 18 gauge needle is usedto detach the collagen gel lattices from the surface of the culturedishes so that they are loose and suspended in saline. A total of 30collagen lattices are prepared to allow quintruplicate measurementsbased on 5 treatment groups plus an untreated control. To form theFPCLs, all saline is aspirated from the 35 mm culture dishes containingthe lattices. Two ml of 2×10̂5 fibroblasts/ml are placed on the surfaceof each of the prefabricated collagen gel lattices. FPCLs are dividedinto six groups as follows: Group I is kept as a control with notreatment; Group II receives nonconditioned media; Group III receivesamnion-derived cell conditioned media; Group IV receives a suspension ofamnion-derived cells; Group V is covered with an extracellular matrix;and Group VI is covered with an extracellular matrix seeded withamnion-derived cells. The FPCLs are incubated at 37° C., 5% carbondioxide. The amount of gel contraction is measured every 24 hours for 5days.

Acetate overlays are used for tracing the area of the gels. Gels areperformed in quintruplicate (5 gels) for the fibroblast line establishedand measurements are calculated using digital planimetry and Sigma Scansoftware (Jandel Scientific, Corte Madera, Calif.). Each collagen gelarea measurement is converted to reflect percentage of area remainingover time and subsequently percentage of gel contraction. A one-wayanalysis of variance is used to determine significant differences amonggroups. When a difference is identified, a Tukey's Test (all pairwisemultiple comparison test) is used to delineate the differences. SigmaStat statistical software is used for data analysis. In addition, the 24hour FPCLs are examined microscopically and photographed. One of thegels is evaluated for fibroblast viability on day 5 utilizing Trypanblue exclusion assay. Another gel is included for cell numberspectrophometrically as a function of mitochondrial activity using theMTT method. After exposure to test agents, the five day suspensioncultures are reincubated and exposed to[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide or MTT.Mitochondrial dehydrogenases from viable fibroblasts cleave thetetrazolium ring, yielding purple formazan crystals. These are dissolvedin an acidified isopropanol resulting in a purple solution which isspectrophometrically measured. An increase or decrease in cell numberresults in a concomitant change in the amount of formazan formed,indicating any degree of toxicity of the applied test material.

Example 20 Differentiation of Amnion-Derived Cells to Early PancreaticProgenitor Cells

All four pancreatic endocrine cell types that comprise the pancreaticislet develop by progressive differentiation from a common pancreaticprogenitor cell. This common pancreatic progenitor cell expresses thePDX1 gene early in its developmental ontogeny and later, expression ofthis gene serves as an early distinguishable cell marker of pancreaticdifferentiation. Once this early pancreatic progenitor cell is generatedand/or propagated in vitro, it may potentially give rise to all fourendocrine cell types comprising the mature pancreatic islet.

Developmentally, the endocrine and exocrine pancreatic cells are derivedfrom outgrowths of cuboidal epithelium from the foregut endoderm. Thisthree-dimensional architecture is thought to be important in thedevelopment and expression of both exocrine and endocrine cell types. Inan attempt to duplicate this environment, a novel culture condition thatpreferentially supports and maintains the spheroids of amnion-derivedcell in suspension culture is used. Briefly, cells are grown as amonolayer, than treated with proteinase XXIII to obtain single cells orsmall clusters of cells. Detection of PDX 1 protein expression(cytoplasmic and peri-nuclear) first occurs in cells present in buds onthe outer-most surface of the amnion-derived cell spheroids.Immunocytochemical techniques show that these cells also express CD29protein. These buds of pancreatic progenitor cells resemble the cellsprominently observed in adult pancreatic duct cell differentiation(Bonner_Weir, S., et al., Proceedings of the National Academy ofSciences of the United States of America, 2000. 97(14): p. 7999-8004.,Jones, E. M. and N. Sarvetnick, Horm Metab Res, 1997. 29(6): p. 308-10;Ramiya, V. K., et al., Nature Medicine, 2000. 6(3): p. 278-82]

The cells are then plated on a substrate or in a matrix that maintainsthe three-dimensional structure of the spheroids but promotes proteinexpression of cytoplasmic PDX1. After several days, the cells aresupplemented with factors that promote nuclear translocation of PDX1protein in a small proportion of the differentiating cells. In thepresence of factor/cell culture conditions that promote nuclearlocalization of PDX1 protein, large numbers of these buds appear on themajority of the amnion-derived cell spheroids cultured in suspension.Amnion-derived cells may also be cultured in the presence of anextracellular matrix in the presence of the above factors. As a result,cells will form spheroids on or embedded in the extracellular matrix.

Example 21 Determination of Cells Expressing Islet Cell Proteins

Cells fixed in 4% buffered PFA and stored in 1×PBS containing 0.02%sodium azide, are rinsed with 1×PBS. The cells are then blocked fornonspecific binding with 5% BSA in Calcium and Magnesium-Free PhosphateBuffered Saline (CMF-PBS) for 30 minutes and permeabilized with PBS-TX(CMF-PBS/0.3% Triton-X-100). Staining for nuclear antigens is performedusing an additional high salt treatment (0.3M NaCl, 20 mM Tris-HCl pH7.2, 0.1% Tween-20, 0.1% Triton-X-100) and overnight incubation with theprimary antibody. All antibodies are diluted in 5% BSA in PBS-TX, unlessotherwise noted. Pancreatic transcription factors are identified bystaining the cells with anti-PDX1 (rabbit polyclonal, 1:2000, C. V.Wright), anti-Nkx2.2 (mouse monoclonal 1:100, T. Jessell), anti-Nkx6.1(mouse monoclonal, 1:8000, T. Jessell), and anti-HB9 (mouse monoclonal,1:30, T. Jessell). Endocrine cells are identified by staining withanti-Insulin (1:2000, Linco 4012-01), anti-Proinsulin, (1:400,Novacastra Peninsula, IHC-7165) and anti-Somatostatin (1:2000,Peninsula, IHC-8001). Secondary antibodies used include: Fluoresceinisothyocyanate (FITC) (1:200), Indocarbocyanine (Cy3) (1:1000) andIndodicarbocyanine (Cy5) (1:400)-conjugated donkey anti-mouse, rabbitand guinea pig IgG (Jackson ImmunoResearch, ML grade). Cell nuclei arevisualized by DAPI fluorescence as part of the Vectashield mountingmedium (Vector, H1200). Cells are analyzed with a Nikon Eclipse E2000 Ufluorescence/DIC inverted microscope equipped with Autoquant 3-D Imagingsoftware and an Olympus FV300 FluoView Confocal Laser ScanningMicroscope.

Example 22 Transplantation Studies In Vivo Co-Culture of Amnion-DerivedCell with Embryonic Islet Progenitor Cells Under the Kidney Capsule

Three experimental groups are used to determine if the dorsal pancreaticbud from embryonic day 12.5 (e12.5) mice promotes the differentiation ofPDX1 protein-expressing amnion-derived cells:

A. Group 1-12 immuno-compromised mice, 4 months of age, are transplantedwith 106 PDX1 protein-expressing cells and two e12.5 dorsal pancreaticexplants under the kidney capsule. Three mice are sacrificed foranalysis every second week (time points: 2, 4, 6, and 8 weeks aftertransplantation).

B. Group 2 (control)-12 immuno-compromised mice, 4 months of age, aretransplanted with 106 freshly isolated amnion-derived cells and twoe12.5 dorsal pancreatic explants under the kidney capsule. Three miceare sacrificed for analysis every second week (time points: 2, 4, 6, and8 weeks after transplantation).

C. Group 3 (control)-12 immuno-compromised mice, 4 months of age, twoe12.5 dorsal pancreatic explants under the kidney capsule. Three miceare sacrificed for analysis every second week (time points: 2, 4, 6, and8 weeks after transplantation).

Dorsal pancreatic buds are manually dissected from the foregut of eachembryo directly and/or incubated with 0.2 Wunsch U/ml LiberaseBlendzyme-3 (Roche, 11814176) and 0.15 mg/ml DNase I at 37° C. for 15min. The enzymatic dissociation is immediately terminated by theaddition of an equal volume of 1×PBS containing 10% (w/v) BSA. Thedorsal pancreatic buds are rinsed with HBSS at 4° C. and brieflytriturated using a 200 μl pipet tip. The epithelium is stripped manuallyfrom the surrounding mesenchyme and transferred into HBSS at 4° C. priorto transplantation.

Mouse and human amnion-derived cells are placed under the kidney capsuleof immuno-compromised mice to prevent immuno-rejection of the graftedcells over the course of the experiment. At each time point, three miceare sacrificed, the transplanted cells isolated and fixed in 4% PFA. Thetissue is then soaked in a series of increasing concentrations ofsucrose, embedded with OCT and sectioned. Cyro-sections (10 μm) areanalyzed for the co-expression of human nuclear antigen protein andspecific islet endocrine cell marker proteins. These markers include:pro-insulin, C-peptide, glucagon, somatostatin, Nkx2.2, Pax6, Nkx6.1 andPDX1 proteins.

Example 23 Transplantation Studies In Vivo Co-Culture of Amnion-DerivedCells with Embryonic Islet Progenitor Cells in the Mammary Gland

Three experimental groups are used to determine if the dorsal pancreaticbud from embryonic day 12.5 (e12.5) mice promotes the differentiation ofPDX1 protein-expressing amnion-derived cells:

A. Group 1-12 immuno-compromised mice, 4 months of age, are transplantedwith 106 PDX1 protein-expressing cells and two e12.5 dorsal pancreaticexplants in the mammary gland. Three mice are sacrificed for analysisevery second week (time points: 2, 4, 6, and 8 weeks aftertransplantation).

B. Group 2 (control)-12 immuno-compromised mice, 4 months of age, aretransplanted with 106 freshly isolated amnion-derived cells and twoe12.5 dorsal pancreatic explants in the mammary gland. Three mice aresacrificed for analysis every second week (time points: 2, 4, 6, and 8weeks after transplantation).

C. Group 3 (control)-12 immuno-compromised mice, 4 months of age, twoe12.5 dorsal pancreatic explants in the mammary gland. Three mice aresacrificed for analysis every second week (time points: 2, 4, 6, and 8weeks after transplantation).

Dorsal pancreatic buds are manually dissected from the foregut of eachembryo directly and/or incubated with 0.2 Wunsch U/ml LiberaseBlendzyme-3 (Roche, 11814176) and 0.15 mg/ml DNase I at 37° C. for 15min. The enzymatic dissociation is immediately terminated by theaddition of an equal volume of 1×PBS containing 10% (w/v) BSA. Thedorsal pancreatic buds are rinsed with HBSS at 4° C. and brieflytriturated using a 200 μl pipet tip. The epithelium is stripped manuallyfrom the surrounding mesenchyme and transferred into HBSS at 4° C. priorto transplantation.

Mouse and human amnion-derived cells are placed in the mammary gland ofimmuno-compromised nude mice to prevent immuno-rejection of the graftedcells over the course of the experiment. At each time point, three miceare sacrificed, the transplanted cells isolated and fixed in 4% PFA. Thetissue is then soaked in a series of increasing concentrations ofsucrose, embedded with OCT and sectioned. Cyro-sections (10 μm) areanalyzed for the co-expression of human nuclear antigen protein andspecific islet endocrine cell marker proteins. These markers include:pro-insulin, C-peptide, glucagon, somatostatin, Nkx2.2, Pax6, Nkx6.1 andPDX1 proteins.

Example 24 Transplantation of Undifferentiated Amnion-Derived Cells,Semi-Differentiated Amnion-Derived Cells Expressing Peri-Nuclear PDX1and Amnion-Derived Cells Stably Expressing the Nuclear PDX1 FusionProtein into Immuno-Comprised Mice

Transplantation of undifferentiated amnion-derived cells,semi-differentiated amnion-derived cells expressing peri-nuclear PDX1and amnion-derived cells stably expressing the nuclear PDX1 fusionprotein into non-diabetic immuno-compromised mice was done to determineif the mammary gland is a permissive transplantation site that willmaintain the morphological characteristics of the differentiating cellsand expression of the PDX1 protein.

Fifteen immuno-compromised mice (Hilltop Labs) were transplanted asfollows: Group 1: Control Group, Reduced Factor Matrigel Injected, 5mice; Group 2: Factor Induced PDX1 expressing amnion-derived cells; 5mice; Group 3: amnion-derived cells infected with Lentiviral PDX1 fusionprotein; 5 mice.

Mice were maintained after transplantation in normal housing conditionsfor 31 days. The mammary gland tissue containing the transplanted cellswas subsequently removed, fixed, embedded with O.C.T. and frozen at −80C. Additional control samples from the opposite (non-transplanted)mammary gland were also excised as a control. Sections will be analyzedfor PDX1, Proinsulin and insulin expression.

Example 25 Further Transplantation Studies

PDX1 protein-expressing cells are also transplanted into the portalvein, spleen and mammary gland using non-diabetic immuno-compromisedmice. Differentiating amnion-derived cells as above are initiallytransplanted with differentiating mouse e12 dorsal pancreatic buds todetermine if they can respond to the same factors as early embryonicpancreatic epithelial cells and generate islet-like cells. All tissuestransplanted with PDX1 protein-expressing cells are removed two and sixweeks following transplantation, fixed, cryopreserved and sectioned.Tissue sections are stained with PDX1 anti-sera, Pro-insulin, C-Peptide,Glucagon and Somatostatin antibodies. Human nuclear antigenimmunostaining will be used to verify the origin of cells expressingendocrine cell markers in the rat pancreas.

Example 26 Restoration of Normoglycemia in STZ-Induced DiabeticNOD-Immun-Compromised Mice

Further experiments are conducted to restore normoglycemia inSTZ-induced diabetic NOD-immuno-compromised mice. Mice exhibitinginitial blood glucose levels over 400 ng/dl are included in theexperiment. Insulin therapy (Linbit) is administered to the animalsafter the initial blood glucose determination but prior to celltransplantation. This allows for the initial engraftment ofamnion-derived cells in a normoglycemic environment. Initial evidence ofhuman C-peptide expression will be determined using a human C-PeptideRIA or ELISA assay. Once detection of human C-peptide is confirmed,insulin therapy is discontinued and the blood glucose monitored everythird day. If the differentiated cells are able to restore normoglycemiain the STZ-induced diabetic mice, the transplanted cells will be removedforty to sixty days after transplantation and the mice evaluated dailyfor reversion to the blood glucose levels (>400 ng/dl) previouslyobserved.

Example 27 Factor-Priming Experiments

In one experiment, the pancreas of a mouse is primed with factors topromote differentiation of resident pancreatic cells into functionalislets. These factors include any individual or combination of thefollowing factors: FGF(s), Forskolin, Follistatin, angiogenic factors,glucocorticoid family members, Insulin, EGF, EGF-like factors, Heparin,Nicotinamide, SHh antagonists, HGF, GLP-1 analogs, between 1 and 20 mMGlucose, divalent cations.

In another experiment the pancreas of a mouse is primed with factors topromote the regeneration of transplanted undifferentiated amnion-derivedcells to this site and to allow differentiation. Undifferentiatedamnion-derived cells may be freshly isolated cells (not cultured)treated with factors to ensure endoderm differentiation (SHhantagonists, spheroid growth).

In another experiment the pancreas of a mouse is primed with factors topromote the regeneration of transplanted partially differentiatedamnion-derived cells to this site and to allow further differentiation.Partially differentiated means the amnion-derived cells have beencultured in vitro in any condition described herein.

In another experiment the pancreas of a mouse is primed with otherfactors and then the following cells are transplanted: Co-culture ofundifferentiated or partially differentiated amnion-derived cells withdifferentiating embryonic pancreatic or non-pancreatic tissue(epithelium, mesenchyme, islets, ducts, exocrine cells) ordifferentiating or pre-differentiated non-embryonic heterologous (donor)or autologous (self) tissue (epithelium, mesenchyme, islets, ducts,exocrine cells, etc.). These cells will provide active factors and/orthe biological niche necessary for the differentiation ofundifferentiated or partially differentiated amnion-derived cells topancreatic cells. These factors and/or niche may also promote themolecular organization of the cells so the cells mature and function aspancreatic islet-like cells.

In another experiment the pancreas is primed with proprietary factorcombinations and then transplanted with a co-culture of undifferentiatedor partially differentiated amnion-derived cells with differentiatingembryonic pancreatic or non-pancreatic tissue (epithelium, mesenchyme,islets, ducts, exocrine cells) or differentiating or pre-differentiatednon-embryonic heterologous (donor) or autologous (self) tissue(epithelium, mesenchyme, islets, ducts, exocrine cells, etc.). Thesecells will provide active factors and/or the biological niche necessaryfor the differentiation of undifferentiated or partially differentiatedamnion-derived cells to pancreatic cells. These factors and/or niche mayalso promote the molecular organization of the cells so the cells matureand function as pancreatic islet-like cells.

Another experiment is transplanting undifferentiated or partiallydifferentiated amnion-derived cells that have been primed in vitro (notprimed at the site of transplantation in vivo) directly into pancreas.In another experiment, the cells are transplanted subcutaneously, intoliver, mammary gland, kidney capsule, spleen or any other site in whichthe cells are able to engraft.

Another experiment is transplanting undifferentiated or partiallydifferentiated amnion-derived cells via intravenous injection topancreas that has been injured surgically or chemically then primed ornot primed with the factors listed above. In this experiment the cellswill “home” to the inflammatory site and integrate with the residentcells.

Another experiment is the use of undifferentiated, partiallydifferentiated or functionally differentiated amnion-derived cellstransplanted into the pancreas or any other tissue (i.e. subcutaneously,into liver, mammary gland, kidney capsule, spleen or any other site inwhich the cells are able to engraft) (or introduced by intravenousinjection) to induce immune tolerance in a patient with an autoimmunedisease (for example, diabetes). Synchronized cell differentiation mayoccur between the transplanted amnion-derived cells alone and/or withcells in the patient (i.e. damaged islets, beta cells, etc). Theamnion-derived cells may provide HLA antigens that will protect cellsassociated with them from the immune system.

Explants will be evaluated for human pancreatic islet progenitor cells.Undifferentiated and partially differentiated amnion-derived cells willdifferentiate into cells expressing islet cell-specific protein markersof differentiation. Analysis will include immunocytochemistry and GFPexpression of pre-labeled cells.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Throughout the specification various publications have been referred to.It is intended that each publication be incorporated by reference in itsentirety into this specification.

1. (canceled)
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 12. A method of obtaining a substantially purified populationof amnion-derived cells, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with (i) one or moreantibodies selected from the group consisting of anti-CD105, anti-CD90,anti-CD117, anti-CD140b, anti-CD34, anti-CD44, and anti-CD45,antibodies; and (ii) one or more antibodies selected from the groupconsisting of anti-CD29, anti-CD9, anti-CD10, anti-CD26, anti-CD71,anti-CD166, anti-CD227, anti-EGF-R, anti-SSEA-4, and anti-HLA-Gantibodies; and c) separating the cells that do not bind to theantibodies of (i) from the cells that do bind to the antibody of (i) andseparating the cells that do not bind to the antibodies of (ii) from thecells that do bind to the antibody of (ii) such that a substantiallypurified population of amnion-derived cells that do not bind to theantibodies of (i) and do bind to the antibody of (ii) is obtained 13.(canceled)
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 16. A composition comprisingconditioned medium obtained from a substantially purified population ofamnion-derived cells that is negative for expression of the proteinmarkers CD90 and CD117.
 17. A composition comprising cell lysateobtained from a substantially purified population of amnion-derivedcells that is negative for expression of the protein markers CD90 andCD117.
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 59. A method of obtaining a substantially purified populationof amnion-derived cells, comprising: a) providing a population ofamnion-derived cells; b) contacting the cells with anti-CD90 andanti-CD117 antibodies; and c) separating the cells that do not bind tothe antibodies of (b) from the cells that do bind to the antibody of (b)such that a substantially purified population of amnion-derived cellsthat do not bind to the antibodies of (b) is obtained.
 60. The method ofclaim 12 wherein the antibodies of (i) are anti-CD105, anti-CD90,anti-CD117, anti-CD140b, anti-CD34, anti-CD44, and anti-CD45 antibodies,and the antibodies of (ii) are anti-CD29, anti-CD9, anti-CD10,anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R, anti-SSEA-4,and anti-HLA-G antibodies.
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 62. The method of claim 59further optionally comprising: d) contacting the cells of c) withanti-HLA-G antibodies; and e) separating the cells that bind to theanti-HLA-G antibodies from the cells that do not bind to the anti-HLA-Gantibodies such that a substantially purified population ofamnion-derived cells that bind to the anti-HLA-G antibodies is obtained.63. The composition of claim 16 wherein the substantially purifiedpopulation of amnion-derived cells is further negative for expression ofthe protein marker CD105.
 64. The composition of claim 16 wherein thesubstantially purified population of amnion-derived cells is positivefor expression of the protein marker CD29.
 65. The composition of claim16 wherein the substantially purified population of amnion-derived cellsis positive for expression of HLA-G.
 66. The composition of claim 16wherein the substantially purified population of amnion-derived cells isfurther negative for the expression of telomerase.
 67. The compositionof claim 66 wherein the substantially purified population ofamnion-derived cells is positive for expression of HLA-G.
 68. Thecomposition of claim 16, which is a pharmaceutical composition.
 69. Thecomposition of claim 16, wherein the amnion-derived cells are anexpanded amnion-derived cell composition.
 70. The composition of claim16, wherein the conditioned medium is diluted with unconditioned medium.71. A method of making a composition comprising conditioned media,comprising a) seeding amnion-derived cells in culture flasks, b)culturing the cells in animal-free medium supplemented with EGF, c)culturing the cells until they reach near confluency, d) removing themedium and applying fresh medium, and e) collecting the freshly appliedmedium after culture.
 72. The method of claim 71 wherein the medium doesnot contain serum.
 73. The method of claim 72 wherein the medium is IMDMmedium.
 74. The method of claim 71 wherein the freshly applied medium ofstep (e) is collected after at least one day in culture.
 75. Acomposition comprising conditioned media made by the method of claim 71.76. A composition comprising conditioned media which is positive forexpression of Angiopoietin-2, Angiogenin, bFGF, EGF, FGF-7, FGF-4,IGF-1, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, PDGF-AA, PDGF-AB,PDGF-BB, PDGF-Ra, and PDGF-Rb as detected by antibody array.
 77. Thecomposition of claim 76 which is negative for the expression of TGF-a,TGF-beta 1, TGF-beta 2, and TGF-beta 3 as detected by antibody array.78. A composition comprising conditioned media obtained from culturedamnion-derived cells.